Dose selection of adjuvanted synthetic nanocarriers

ABSTRACT

Disclosed are synthetic nanocarrier compositions with coupled adjuvant compositions as well as related methods.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/717,451, filed May 20, 2015, now allowed, which is a continuation ofU.S. patent application Ser. No. 13/116,542, filed May 26, 2011, nowgranted as U.S. Pat. No. 9,066,978, which claims the benefit under 35U.S.C. § 119 of U.S. Provisional Application Nos. 61/348,713 filed May26, 2010, 61/348,717 filed May 26, 2010, 61/348,728, filed May 26, 2010,and 61/358,635, filed Jun. 25, 2010, the entire contents of each ofwhich are incorporated herein by reference.

SEQUENCE LISTING

The contents of the ASCII text file entitled “S168170015US03-SEQ-JAV”,created on Aug. 23, 2017 and 1 kb in size, is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Adjuvants are important components of the majority of currently usedvaccination regimens. They are likely to be integrated into futurevaccine products as well. Numerous novel adjuvants are now beingdeveloped, and many of those have been demonstrated to augment immuneresponses to vaccines in research and clinical settings. However,adjuvant doses that are beneficial for immune response augmentation canbe capable of inducing side-effects in a significant group of patients.In fact, these two capacities of adjuvants are intrinsically linkedsince it is the broad immune stimulation per se that provides stimulifor vaccination augmentation as well as its side-effects (toxicities).Both of these processes are known to be driven by release ofinflammatory cytokines. Therefore, approaches that diminish side-effectsof adjuvant administration and/or specifically augment certain immuneresponses, will be of great clinical value.

Therefore, what is needed are compositions and methods that effectivelyprovide desired immune response(s) that can reduce the frequency ofadverse events associated with adjuvant use in vaccines.

SUMMARY OF THE INVENTION

In one aspect, a method comprising providing a dose of adjuvant and adose of antigen, wherein at least a portion of the dose of adjuvant iscoupled to synthetic nanocarriers, and generating an antibody titeragainst the antigen through administration of the dose of adjuvant andthe dose of antigen to a subject, wherein the dose of adjuvant is lessthan a separate dose of adjuvant that results in an antibody titersimilar to that generated through administration of the dose of adjuvantand the dose of antigen to the subject is provided. In one embodiment,the method further comprises choosing the dose of adjuvant to be lessthan a separate dose of adjuvant that results in an antibody titersimilar to that generated through administration of the dose of adjuvantand the dose of antigen to the subject. Preferably, the same entityperforms each of the steps of these methods (i.e., the same entityperforms the providing, generating and/or choosing steps). In anotheraspect, a composition comprising the dose of adjuvant that is less thana separate dose of adjuvant that results in an antibody titer similar tothat generated through administration of the dose of adjuvant and thedose of antigen to the subject is provided.

In another aspect, a method comprising providing a dose of adjuvant,wherein at least a portion of the dose of adjuvant is coupled tosynthetic nanocarriers, and generating a systemic cytokine releasethrough administration of the dose of adjuvant to a subject, wherein thedose of adjuvant is greater than a separate dose of adjuvant thatresults in a systemic cytokine release similar to that generated throughadministration of the dose of adjuvant to the subject is provided. Inone embodiment, the method further comprises choosing the dose ofadjuvant to be greater than a separate dose of adjuvant that results ina systemic cytokine release similar to that generated throughadministration of the dose of adjuvant to the subject. Preferably, thesame entity performs each of the steps of these methods (i.e., the sameentity performs the providing, generating and/or choosing steps). Inanother aspect, a composition comprising the dose of adjuvant that isgreater than a separate dose of adjuvant that results in a systemiccytokine release similar to that generated through administration of thedose of adjuvant to the subject is provided.

In one embodiment, the adjuvant(s) of any of the methods andcompositions provided herein comprise an agonist for Toll-Like Receptors3, 4, 5, 7, 8, or 9 or a combination thereof. In another embodiment, theadjuvant comprises an agonist for Toll-Like Receptors 3, an agonist forToll-Like Receptors 7 and 8, or an agonist for Toll-Like Receptor 9. Inyet another embodiment, the adjuvant comprises R848, immunostimulatoryDNA, or immunostimulatory RNA. In a further embodiment, the dose ofadjuvant of any of the methods and compositions provided hereincomprises two or more types of adjuvants. In one embodiment, a portionof the dose of adjuvant is not coupled to the synthetic nanocarriers.

In another embodiment of any of the methods and compositions providedherein more than one type of antigen are administered to the subject. Inone embodiment, at least a portion of the dose of antigen(s) is coupledto the synthetic nanocarriers. In another embodiment, at least a portionof the dose of antigen(s) is not coupled to the synthetic nanocarriers.In yet another embodiment, at least a portion of the dose of antigen(s)is coadministered with the synthetic nanocarriers. In still anotherembodiment, at least a portion of the dose of antigen(s) is notcoadministered with the synthetic nanocarriers. In one embodiment, theantigen(s) comprise a B cell antigen and/or a T cell antigen. In anotherembodiment, the T cell antigen comprises a universal T cell antigen orT-helper cell antigen. In still another embodiment, the antigen(s)comprise a B cell antigen or a T cell antigen and a universal T cellantigen or T-helper cell antigen. In one embodiment, the T helper cellantigen comprises a peptide obtained or derived from ovalbumin. Inanother embodiment, the peptide obtained or derived from ovalbumincomprises the sequence as set forth in SEQ ID NO: 1. In still anotherembodiment of any of the methods and compositions provided herein, theuniversal T cell antigen or T helper cell antigen is coupled to thesynthetic nanocarriers by encapsulation. In yet another embodiment ofany of the methods and compositions provided herein, the B cell antigencomprises nicotine. In a further embodiment, the synthetic nanocarrierscomprise nicotine and a universal T cell antigen or T helper cellantigen. In still a further embodiment, the nicotine and/or universal Tcell antigen or T helper cell antigen are coupled to the syntheticnanocarriers. In one embodiment, the universal T cell antigen or Thelper cell antigen is coupled by encapsulation.

In another embodiment of any of the methods and compositions provided,the dose of adjuvant comprises R848 and the dose of antigen comprisesnicotine and a universal T cell antigen or T-helper cell antigen,wherein the nicotine and universal T cell antigen or T-helper cellantigen are also coupled to the synthetic nanocarriers, and wherein thesynthetic nanocarriers comprise one or more polymers.

In another embodiment of any of the methods and compositions providedherein, the synthetic nanocarriers comprise lipid nanoparticles,polymeric nanoparticles, metallic nanoparticles, surfactant-basedemulsions, dendrimers, buckyballs, nanowires, virus-like particles,peptide or protein particles, nanoparticles that comprise a combinationof nanomaterials, spheroidal nanoparticles, cuboidal nanoparticles,pyramidal nanoparticles, oblong nanoparticles, cylindricalnanoparticles, or toroidal nanoparticles. In one embodiment, thesynthetic nanocarriers comprise one or more polymers. In anotherembodiment, the one or more polymers comprise a polyester. In yetanother embodiment, the one or more polymers comprise or furthercomprise a polyester coupled to a hydrophilic polymer. In still anotherembodiment, the polyester comprises a poly(lactic acid), poly(glycolicacid), poly(lactic-co-glycolic acid), or polycaprolactone. In oneembodiment, the hydrophilic polymer comprises a polyether. In anotherembodiment, the polyether comprises polyethylene glycol.

In one embodiment of any of the methods and compositions provided, atleast one dosage form comprises the dose of adjuvant. In anotherembodiment, a vaccine comprises the dosage form(s). In still anotherembodiment, the more than one dosage form comprise the dose of adjuvant,and the more than one dosage form are co-administered.

In one embodiment of any of the methods provided, the administration isby a route that comprises subcutaneous, intramuscular, intradermal,oral, intranasal, transmucosal, rectal; ophthalmic, transdermal ortranscutaneous administration, or a combination thereof.

In another embodiment of any of the methods provided, the subject hascancer, an infectious disease, a non-autoimmune metabolic disease, adegenerative disease, an addiction, and atopic condition, asthma;chronic obstructive pulmonary disease (COPD) or a chronic infection.

In another aspect, a dose of adjuvant and dose of antigen or dose ofadjuvant, as defined in regard to any of the methods or compositionsprovided, for use in therapy or prophylaxis is provided.

In yet another aspect, a dose of adjuvant and dose of antigen or dose ofadjuvant, as defined in regard to any of the methods or compositionsprovided, for use in any of the methods provided is provided.

In still another aspect, a dose of adjuvant and dose of antigen or doseof adjuvant, as defined in regard to any of the methods or compositionsprovided, for use in a method of treating cancer, an infectious disease,a non-autoimmune metabolic disease, a degenerative disease, anaddiction, and atopic condition, asthma; chronic obstructive pulmonarydisease (COPD) or a chronic infection is provided. In one embodiment,the method comprises administration of the dose(s) by a route thatcomprises subcutaneous, intramuscular, intradermal, oral, intranasal,transmucosal, rectal; ophthalmic, transdermal or transcutaneousadministration, or a combination thereof.

In a further aspect, a use of a dose of adjuvant and dose of antigen ordose of adjuvant as defined in regard to any of the methods orcompositions provided, for the manufacture of a medicament for use inany of the methods provided is provided.

BRIEF DESCRIPTION OF FIGURES

FIGS. A, 1B, and 1C show the systemic cytokine production in mice afternanocarrier (NC) inoculation, the production of TNF-α, IL-6, and IL-12in experimental groups, respectively. Sera from groups of three micewere pooled and analyzed by ELISA.

FIG. 2 shows the systemic IFN-γ production in mice after NC inoculation.Sera from groups of three mice were pooled and analyzed by ELISA.

FIG. 3 shows the systemic IL-12 production in mice after inoculationwith free or NC-coupled TLR agonists. Sera from groups of two mice werepooled and analyzed by ELISA.

FIG. 4 shows the local induction of immune cytokines by free orNC-coupled TLR agoinsts. Each point represents an average of two lymphnodes (LNs) from separate mice.

FIG. 5 shows the cell population dynamics in popliteal lymph nodes afterinoculation with free and NC-coupled TLR7/8 agonist R848. Three intactmice were sacrificed at different days and the average cell counts fromtheir popliteal LN assigned “day 0” meaning of “1” to which all othernumbers were compared. Each bar from R848- or NC-inoculated grouprepresents an average from two lymph nodes taken from independentanimals.

FIG. 6 shows anti-nicotine antibody titers in mice immunized with NCcontaining surface nicotine and T-helper peptide OP-II with or withoutR848.

FIGS. 7A and 7B shows that TNF-α and IL-6 were induced in sera ofNC-CpG- and free CpG-inoculated animals.

FIG. 8 shows the induction of IFN-γ and IL-12 in sera of NC-CpG- andfree CpG-inoculated animals.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a polymer”includes a mixture of two or more such molecules, reference to “asolvent” includes a mixture of two or more such solvents, reference to“an adhesive” includes mixtures of two or more such materials, and thelike.

Introduction

The inventors have unexpectedly and surprisingly discovered that theproblems and limitations noted above can be overcome by practicing theinvention disclosed herein. The discoveries described herein relate toadjuvant coupling to nanocarriers, and based on these discoveriesmethods and related compositions are provided that are directed togenerating desired immune responses through the selection of specificdoses of adjuvant coupled to nanocarriers. In some embodiments, anddepending on the desired immune response(s), these doses are less thandoses of adjuvant not coupled to nanocarriers in a similar context. Inother embodiments, these doses are greater than doses of adjuvant notcoupled to nanocarriers.

In one aspect, the inventors have unexpectedly discovered that it ispossible to provide methods, and related compositions, that comprise amethod comprising administering a dose of adjuvant, when coupled tosynthetic nanocarriers, that is less than a separate dose of adjuvantthat results in an immune response (e.g., antibody titer) similar tothat generated through administration of the dose of adjuvant to asubject. Because of the stronger adjuvant effect as a result of couplingat least a portion of a dose of adjuvant to a synthetic nanocarrier,less adjuvant may be used. The doses of adjuvant, therefore, can besub-therapeutic or toxicity-reduced doses, wherein at least a portion ofthe dose of the adjuvant is coupled to synthetic nanocarriers. Inanother aspect, the invention relates to a composition comprising adosage form comprising a sub-therapeutic or toxicity-reduced dose ofadjuvant, and a pharmaceutically acceptable excipient, wherein at leasta portion of the dose of the adjuvant is coupled to syntheticnanocarriers. In still another aspect, the invention relations to amethod comprising administering a sub-therapeutic or toxicity-reduceddose of adjuvant to a subject; wherein at least a portion of the dose ofthe adjuvant is coupled to synthetic nanocarriers.

Coupling of adjuvants to nanocarriers was observed to provide a strongeradjuvant effect and to lead to a substantially higher antibody responsewhen compared to admixed adjuvant. In addition, it was also observedthat coupled adjuvant results in a greater antibody response even when asubstantially greater amount of free adjuvant (as much as 6-foldgreater) is used. See Example 11. This result is contrary to what isexpected from the teachings provided in Diwan et al., Current DrugDelivery, 2004, 1, 405-412, where it was found that antibody production,particularly at lower doses of adjuvant, was higher when adjuvant wasgiven in solution rather than with particulate delivery. An oppositeresult, however, is described herein.

In another aspect, therefore, the inventors have unexpectedly discoveredthat it is possible to provide methods, and related compositions, thatcomprise a method comprising providing a dose of adjuvant and a dose ofantigen, wherein at least a portion of the dose of adjuvant is coupledto synthetic nanocarriers, and generating an antibody titer against theantigen through administration of the dose of adjuvant and the dose ofantigen to a subject, wherein the dose of adjuvant is less than aseparate dose of adjuvant that results in an antibody titer similar tothat generated through administration of the dose of adjuvant and thedose of antigen to the subject. In embodiments, the method furthercomprises choosing the dose of adjuvant to be less than a separate doseof adjuvant that results in an antibody titer similar to that generatedthrough administration of the dose of adjuvant and the dose of antigento the subject (e.g., a human). Preferably, the steps of the methodsprovided herein are performed by the same entity. In still anotheraspect, the invention relates to a composition comprising a dosage formcomprising a dose of adjuvant and a dose of antigen and apharmaceutically acceptable excipient, wherein at least a portion of thedose of adjuvant is coupled to synthetic nanocarriers, and wherein thedose of adjuvant is less than a separate dose of adjuvant that resultsin an antibody titer similar to that generated through administration ofthe dose of adjuvant and the dose of antigen to a subject.

It has also been demonstrated that coupling of adjuvant to nanocarrierscan result in a lower immediate systemic cytokine induction thanutilizing free adjuvant. Therefore, coupling of adjuvant to nanocarrierscan allow for the use of a higher dose of adjuvant as compared toseparate adjuvant. In another aspect, therefore, the invention relatesto a method comprising providing a dose of adjuvant, wherein at least aportion of the dose of adjuvant is coupled to synthetic nanocarriers,generating an immune response (e.g., a systemic cytokine release)through administration of the dose of adjuvant to a subject (e.g., ahuman), wherein the dose of adjuvant is greater than a separate dose ofadjuvant that results in the immune response similar to that generatedthrough administration of the dose of adjuvant to the subject. Inembodiments, the method further comprises choosing the dose of adjuvantto be greater than a separate dose of adjuvant that results in an immuneresponse (e.g., systemic cytokine release) similar to that generatedthrough administration of the dose of adjuvant to the subject.Preferably, the steps of the methods provided herein are performed bythe same entity. In yet another aspect, the invention relates to acomposition comprising a dosage form comprising a dose of adjuvant and apharmaceutically acceptable excipient, wherein at least a portion of thedose of the adjuvant is coupled to synthetic nanocarriers, and whereinthe dose of adjuvant is greater than a separate dose of adjuvant thatresults in an immune response (e.g., systemic cytokine release) similarto that generated through administration of the dose of adjuvant to thesubject.

Collectively, with the discoveries provided herein it is now possible toselect an adjuvant dose depending on the desired immune result that isspecific for the use of adjuvant coupled to nanocarriers. The dose canbe a lower one (as compared to separate adjuvant) that generatesantibody titers or that avoids unwanted systemic activity (whilestrongly potentiating local immunostimulatory effects). The dose can bea greater one that generates a similar systemic cytokine release profileas compared to separate adjuvant.

In a further aspect, the administration of compositions provided hereincan be beneficial to any subject in which the modulation of an immuneresponse is desired. In some embodiments, the subject is one in which aninflammatory response is desired. In other embodiments, the subjects arethose where a Th1 immune response is desired. In some embodiments, thesubjects have or are at risk of having cancer. In other embodiments, thesubjects have or are at risk of having an infection or an infectiousdisease. In still other embodiments, the subjects have or are at risk ofhaving an atopic condition, asthma, chronic obstructive pulmonarydisease (COPD) or a chronic infection. Methods for the administration ofthe compositions to such subjects are also provided.

Examples 1-13 illustrates various embodiments of the present invention,including different formulations or aspects of the present invention.The compositions and methods described in the Examples are also providedherein.

The invention will now be described in more detail below.

Definitions

“Adjuvant” means an agent that does not constitute a specific antigen,but boosts the strength and longevity of immune response to aconcomitantly administered antigen. Such adjuvants may include, but arenot limited to stimulators of pattern recognition receptors, such asToll-like receptors, RIG-1 and NOD-like receptors (NLR), mineral salts,such as alum, alum combined with monphosphoryl lipid (MPL) A ofEnterobacteria, such as Escherihia coli, Salmonella minnesota,Salmonella typhimurium, or Shigella flexneri or specifically with MPL®(AS04), MPL A of above-mentioned bacteria separately, saponins, such asQS-21, Quil-A, ISCOMs, ISCOMATRIX™, emulsions such as MF59™, Montanide®ISA 51 and ISA 720, AS02 (QS21+squalene+MPL®), AS15, liposomes andliposomal formulations such as AS01, synthesized or specificallyprepared microparticles and microcarriers such as bacteria-derived outermembrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis andothers, or chitosan particles, depot-forming agents, such as Pluronic®block co-polymers, specifically modified or prepared peptides, such asmuramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529,or proteins, such as bacterial toxoids or toxin fragments.

In embodiments, adjuvants comprise agonists for pattern recognitionreceptors (PRR), including, but not limited to Toll-Like Receptors(TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/or combinationsthereof. In other embodiments, adjuvants comprise agonists for Toll-LikeReceptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists forToll-Like Receptor 9; preferably the recited adjuvants compriseimidazoquinolines; such as R848; adenine derivatives, such as thosedisclosed in U.S. Pat. No. 6,329,381 (Sumitomo Pharmaceutical Company),US Published Patent Application 2010/0075995 to Biggadike et al., or WO2010/018132 to Campos et al.; immunostimulatory DNA; orimmunostimulatory RNA. In specific embodiments, synthetic nanocarriersincorporate as adjuvants compounds that are agonists for toll-likereceptors (TLRs) 7 & 8 (“TLR 7/8 agonists”). Of utility are the TLR 7/8agonist compounds disclosed in U.S. Pat. No. 6,696,076 to Tomai et al.,including but not limited to imidazoquinoline amines, imidazopyridineamines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2-bridgedimidazoquinoline amines. Preferred adjuvants comprise imiquimod andresiquimod (also known as R848). In specific embodiments, an adjuvantmay be an agonist for the DC surface molecule CD40. In certainembodiments, to stimulate immunity rather than tolerance, a syntheticnanocarrier incorporates an adjuvant that promotes DC maturation (neededfor priming of naive T cells) and the production of cytokines, such astype I interferons, which promote antibody immune responses. Inembodiments, adjuvants also may comprise immunostimulatory RNAmolecules, such as but not limited to dsRNA, poly I:C or poly I:poly C12U (available as Ampligen®, both poly I:C and poly I:polyC12U beingknown as TLR3 stimulants), and/or those disclosed in F. Heil et al.,“Species-Specific Recognition of Single-Stranded RNA via Toll-likeReceptor 7 and 8” Science 303(5663), 1526-1529 (2004); J. Vollmer etal., “Immune modulation by chemically modified ribonucleosides andoligoribonucleotides” WO 2008033432 A2; A. Forsbach et al.,“Immunostimulatory oligoribonucleotides containing specific sequencemotif(s) and targeting the Toll-like receptor 8 pathway” WO 2007062107A2; E. Uhlmann et al., “Modified oligoribonucleotide analogs withenhanced immunostimulatory activity” U.S. Pat. Appl. Publ. US2006241076; G. Lipford et al., “Immunostimulatory viral RNAoligonucleotides and use for treating cancer and infections” WO2005097993 A2; G. Lipford et al., “Immunostimulatory G,U-containingoligoribonucleotides, compositions, and screening methods” WO 2003086280A2. In some embodiments, an adjuvant may be a TLR-4 agonist, such asbacterial lipopolysacccharide (LPS), VSV-G, and/or HMGB-1. In someembodiments, adjuvants may comprise TLR-5 agonists, such as flagellin,or portions or derivatives thereof, including but not limited to thosedisclosed in U.S. Pat. Nos. 6,130,082, 6,585,980, and 7,192,725. Inspecific embodiments, synthetic nanocarriers incorporate a ligand forToll-like receptor (TLR)-9, such as immunostimulatory DNA moleculescomprising CpGs, which induce type I interferon secretion, and stimulateT and B cell activation leading to increased antibody production andcytotoxic T cell responses (Krieg et al., CpG motifs in bacterial DNAtrigger direct B cell activation. Nature. 1995. 374:546-549; Chu et al.CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1(Th1) immunity. J. Exp. Med. 1997. 186:1623-1631; Lipford et al.CpG-containing synthetic oligonucleotides promote B and cytotoxic T cellresponses to protein antigen: a new class of vaccine adjuvants. Eur. J.Immunol. 1997. 27:2340-2344; Roman et al. Immunostimulatory DNAsequences function as T helper-1-promoting adjuvants. . Nat. Med. 1997.3:849-854; Davis et al. CpG DNA is a potent enhancer of specificimmunity in mice immunized with recombinant hepatitis B surface antigen.J. Immunol. 1998. 160:870-876; Lipford et al., Bacterial DNA as immunecell activator. Trends Microbiol. 1998. 6:496-500; U.S. Pat. No.6,207,646 to Krieg et al.; U.S. Pat. No. 7,223,398 to Tuck et al.; U.S.Pat. No. 7,250,403 to Van Nest et al.; or U.S. Pat. No. 7,566,703 toKrieg et al.

In some embodiments, adjuvants may be proinflammatory stimuli releasedfrom necrotic cells (e.g., urate crystals). In some embodiments,adjuvants may be activated components of the complement cascade (e.g.,CD21, CD35, etc.). In some embodiments, adjuvants may be activatedcomponents of immune complexes. The adjuvants also include complementreceptor agonists, such as a molecule that binds to CD21 or CD35. Insome embodiments, the complement receptor agonist induces endogenouscomplement opsonization of the synthetic nanocarrier. In someembodiments, adjuvants are cytokines, which are small proteins orbiological factors (in the range of 5 kD-20 kD) that are released bycells and have specific effects on cell-cell interaction, communicationand behavior of other cells. In some embodiments, the cytokine receptoragonist is a small molecule, antibody, fusion protein, or aptamer.

In embodiments, at least a portion of the dose of adjuvant is coupled tosynthetic nanocarriers, preferably, all of the dose of adjuvant iscoupled to synthetic nanocarriers. In embodiments, the dose of adjuvantcomprises two or more types of adjuvants. For instance, and withoutlimitation, adjuvants that act on different receptors, such as differentTLR receptors may be combined. As an example, in an embodiment a TLR 7/8agonist may be combined with a TLR 9 agonist. In another embodiment, aTLR 7/8 agonist may be combined with a TLR 4 agonist. In yet anotherembodiment, a TLR 9 agonist may be combined with a TLR 3 agonist.

“Administering” or “administration” means providing a substance to asubject in a manner that is pharmacologically useful.

“Amount effective” is any amount of a composition that produces one ormore desired immune responses. This amount can be for in vitro or invivo purposes. For in vivo purposes, the amount can be one that aclinician would believe may have a clinical benefit for a subject inneed of an immune response. Such subjects include those that have or areat risk of having cancer, an infection or infectious disease, an atopiccondition, asthma, chronic obstructive pulmonary disease (COPD) or achronic infection.

Amounts effective include those that involve the generation of anantibody titer and/or the systemic release of one or more cytokines. Inembodiments, the amounts effective include those that involve theproduction of a systemic cytokine release profile. In some embodiments,the one or more cytokines or cytokine release profile comprises thesystemic release of TNF-α, IL-6 and/or IL-12. In other embodiments, theone or more cytokines or cytokine release profile comprises the systemicrelease of IFN-γ, IL-12 and/or IL-18. This can be monitored by routinemethods. An amount that is effective to produce one or more desiredimmune responses can also be an amount of a composition provided hereinthat produces a desired therapeutic endpoint or a desired therapeuticresult.

Amounts effective will depend, of course, on the particular subjectbeing treated; the severity of a condition, disease or disorder; theindividual patient parameters including age, physical condition, sizeand weight; the duration of the treatment; the nature of concurrenttherapy (if any); the specific route of administration and like factorswithin the knowledge and expertise of the health practitioner. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is generallypreferred that a “maximum dose” be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons.

In embodiments, the selection of the doses of adjuvant(s) coupled tonanocarriers is dependent on a comparison with doses of separateadjuvant(s) (i.e., not coupled to nanocarriers) that generate a similarimmune response (with or without antigen). As used herein, a “similarimmune response” includes immune responses that a health practitionerwould expect to result in a comparable therapeutic result in a subject.Similar immune responses also include immune responses that are the sametype of response (e.g., the induction of the same particular cytokine orset of cytokines, the generation of the same type of antibody titer,etc.), the level of which is not considered to be statisticallydifferent.

Whether or not a similar immune response is generated can be determinedwith in vitro or in vivo techniques. For example, whether or not asimilar immune response is generated can be determined by measuring animmune response (e.g., antibody titer or cytokine(s) release) in asubject through the administration of the dose of separate adjuvant(with or without antigen) to the subject. The subject is not necessarilythe same subject to which the inventive composition comprisingnanocarrier coupled adjuvant are administered in the inventive methods.The subject, for example, can be a clinical trial subject or subjects towhich the dose of separate adjuvant was previously administered. Thesubject can also be an animal model subject or subjects to which thedose of separate adjuvant was previously administered. The determinationof the immune response in the subject can also be determined bymeasuring the response of cells isolated from the subject, or cells fromanother subject or subjects, that are placed in contact with the dose ofseparate adjuvant (with or without antigen). The other subject orsubjects again can be previous clinical trial subjects or animal modelsubjects.

In embodiments, the comparison is based on the measurement of an immuneresponse (e.g., particular type of antibody titer, particular cytokinelevel, levels of a set of cytokines) can be done within the first 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40 or more hoursafter immunization with the dose of separate adjuvant. In otherembodiments, the immune response is measured within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 15, 20, 25, 30, 35, 40 or more days after immunization.Assays for determining whether or not an immune response is similar areknown to those of ordinary skill in the art. Additionally, examples ofsuch assays are described in more detail in the Examples.

Whether or not a dose of separate adjuvant (with or without antigen)generates a similar immune response can also be determined by what ahealth practitioner would expect the immune response (or level of immuneresponse) to be based on results from prior in vitro and/or in vivoassays (in other subjects). Such results can include results fromclinical trials where effective doses have been determined. Accordingly,the dose of separate adjuvant that is used in the comparison is anamount a health practitioner would expect to be effective to produce theimmune response or therapeutic effect. In another embodiment, the doseof separate adjuvant that is used in the comparison is the dose ofseparate adjuvant a health practitioner would expect to be the maximumtolerated dose. In embodiments, the dose of coupled adjuvant is 1-fold,2-fold, 3-fold, 4-fold, 5-fold or 6-fold less than a dose of separateadjuvant that is an amount effective to generate an immune response ortherapeutic result provided herein. In other embodiments, the dose ofcoupled adjuvant is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold or6-fold less than a dose of separate adjuvant that is a maximum tolerateddose. In other embodiments, the dose of coupled adjuvants is greaterthan a dose of separate adjuvant that is an amount effective to generatean immune response or therapeutic result provided herein. In otherembodiments, the dose of coupled adjuvant is greater than a dose ofseparate adjuvant that is a maximum tolerated dose.

In general, doses of the adjuvant(s) or antigen(s) of the compositionsof the invention can range from about 0.001 μg/kg to about 100 mg/kg. Insome embodiments, the doses can range from about 0.01 μg/kg to about 10mg/kg. In still other embodiments, the doses can range from about 0.1μg/kg to about 5 mg/kg, about 1 μg/kg to about 1 mg/kg, about 10 μg/kgto about 0.5 mg/kg or about 100 μg/kg to about 0.5 mg/kg. In furtherembodiments, the doses can range from about 0.1 μg/kg to about 100μg/kg. In still further embodiments, the doses can range from about 30μg/kg to about 300 μg/kg. Alternatively, the dose can be administeredbased on the number of synthetic nanocarriers. For example, useful dosesinclude greater than 10⁶, 10⁷, 10⁸, 10⁹ or 10¹⁰ synthetic nanocarriersper dose. Other examples of useful doses include from about 1×10⁶ toabout 1×10¹⁰, about 1×10⁷ to about 1×10⁹ or about 1×10⁸ to about 1×10⁹synthetic nanocarriers per dose.

In embodiments, the dose is a “sub-therapeutic dose”, which means anamount (e.g. specified number of mass units) of an adjuvant (oradjuvants) that provides a desired therapeutic outcome wherein thesub-therapeutic dose is an amount which is numerically less than wouldbe required to provide substantially the same therapeutic outcome ifadministered separately. In this context, “separate” or “separately”means that adjuvant (or adjuvants) is not coupled to a syntheticnanocarrier. In an embodiment, the sub-therapeutic dose of R848comprises from 0.01 micrograms/kg to 100 micrograms/kg, preferably 0.1micrograms/kg to 10 micrograms/kg, of R848. In an embodiment, thesub-therapeutic dose of CpG containing oligonucleotide comprises from0.001 μg/kg to 2 mg/kg, preferably from about 0.01 μg/kg to 0.1 mg/kg ,of CpG containing oligonucleotide. In yet another embodiment, thesub-therapeutic dose of an immunologically active nucleic acid or aderivative thereof comprises from 0.001 μg/kg to 2 mg/kg, preferablyfrom 0.01 ∥g/kg to 0.1 mg/kg. In another embodiment, a sub-therapeuticdose of MPL® comprises from 0.001 μg/kg to 0.5 mg/kg.

In other embodiments, the dose is a “toxicity-reduced dose”, which meansa dose of an adjuvant that provides a particular systemic cytokinerelease, preferably a particular systemic cytokine release profile,wherein the toxicity-reduced dose is greater than a dose of adjuvantthat would be required to provide substantially the same particularsystemic cytokine release, preferably a particular systemic cytokinerelease profile, when administered separately. In this context,“separately” means adjuvant that is not coupled to a syntheticnanocarrier. Additionally, “systemic cytokine release profile” means apattern of systemic cytokine release, wherein the pattern comprisescytokine levels measured for several different systemic cytokines. In anembodiment, the toxicity-reduced dose of R848 comprises from 0.01micrograms/kg to 100 micrograms/kg, preferably 0.1 micrograms/kg to 10micrograms/kg, of R848. In an embodiment, the toxicity-reduced dose ofCpG containing oligonucleotide comprises from 0.001 micrograms/kg to 2mg/kg, preferably 0.01 μg/kg micrograms to 0.1 mg/kg, of CpG containingoligonucleotide. In another embodiment, sub-therapeutic dose of MPL®comprises from 0.001 μg/kg to 0.5 mg/kg.

“Antibody response” means any immune response that results in theproduction or stimulation of B cells and/or the production ofantibodies. “Antibody titer” means the production of a measurable levelof antibodies. Preferably, the antibody response or generation of theantibody titer is in a human. In some embodiments, the antibodies areantibodies of a certain isotype, such as IgG or a subclass thereof.Methods for measuring antibody titers are known in the art and includeEnzyme-linked Immunosorbent Assay (ELISA). Methods for measuringantibody titers are also described in some detail in the Examples.Preferably, the antibody response or antibody titer is specific to anantigen. Such antigen can be coadministered with the adjuvant couplednanocarrier but can also not be coadministered.

“Antigen” means a B cell antigen or T cell antigen. In embodiments,antigens are coupled to the synthetic nanocarriers. In otherembodiments, antigens are not coupled to the synthetic nanocarriers. Inembodiments antigens are coadministered with the synthetic nanocarriers.In other embodiments antigens are not coadministered with the syntheticnanocarriers. “Type(s) of antigens” means molecules that share the same,or substantially the same, antigenic characteristics. In embodiments,antigens of the compositions provided are associated with the disease orcondition that is being treated. For example, the antigen can be anallergen (for the treatment of an allergy or allergic condition), acancer-associated antigen (for the treatment of cancer or a tumor), aninfectious agent antigen (for the treatment of an infection, aninfectious disease or a chronic infectious disease), etc.

“At least a portion of the dose” means at least some part of the dose,ranging up to including all of the dose.

An “at risk” subject is one in which a health practitioner believes hasa chance of having a disease or condition as provided herein.

“B cell antigen” means any antigen that is recognized by a B cell, andtriggers an immune response in a B cell (e.g., an antigen that isspecifically recognized by a B cell receptor on a B cell). In someembodiments, an antigen that is a T cell antigen is also a B cellantigen. In other embodiments, the T cell antigen is not also a B cellantigen. B cell antigens include, but are not limited to proteins,peptides, small molecules, and carbohydrates. In some embodiments, the Bcell antigen comprises a non-protein antigen (i.e., not a protein orpeptide antigen). In some embodiments, the B cell antigen comprises acarbohydrate associated with an infectious agent. In some embodiments,the B cell antigen comprises a glycoprotein or glycopeptide associatedwith an infectious agent. The infectious agent can be a bacterium,virus, fungus, protozoan, parasite or prion. In some embodiments, the Bcell antigen comprises a poorly immunogenic antigen. In someembodiments, the B cell antigen comprises an abused substance or aportion thereof. In some embodiments, the B cell antigen comprises anaddictive substance or a portion thereof. Addictive substances include,but are not limited to, nicotine, a narcotic, a cough suppressant, atranquilizer, and a sedative. In some embodiments, the B cell antigencomprises a toxin, such as a toxin from a chemical weapon or naturalsource, or a pollutant. The B cell antigen may also comprise a hazardousenvironmental agent. In other embodiments, the B cell antigen comprisesan alloantigen, an allergen, a contact sensitizer, a degenerativedisease antigen, a hapten, an infectious disease antigen, a cancerantigen, an atopic disease antigen, an autoimmune disease antigen, anaddictive substance, a xenoantigen, or a metabolic disease enzyme orenzymatic product thereof.

“Choosing” means making a selection either directly oneself orindirectly, such as, but not limited to, an unrelated third party thattakes an action through reliance on one's words or deeds. Generally, thesame entity (e.g., individual, group of individuals acting in concert,or organization) provides a composition provided herein and generatesthe desired immune response through administration of the compositionafter also selecting the appropriate dose of the composition.

“Coadministered” means administering two or more substances to a subjectin a manner that is correlated in time, preferably sufficientlycorrelated in time so as to provide a modulation in an immune response.In embodiments, coadministration may occur through administration of twoor more substances in the same dosage form. In other embodiments,coadministration may encompass administration of two or more substancesin different dosage forms, but within a specified period of time,preferably within 1 month, more preferably within 1 week, still morepreferably within 1 day, and even more preferably within 1 hour.

“Couple” or “Coupled” or “Couples” (and the like) means to chemicallyassociate one entity (for example a moiety) with another. In someembodiments, the coupling is covalent, meaning that the coupling occursin the context of the presence of a covalent bond between the twoentities. In non-covalent embodiments, the non-covalent coupling ismediated by non-covalent interactions including but not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. In embodiments,encapsulation is a form of coupling. In embodiments, at least a portionof a dose of adjuvant(s) is coupled to synthetic nanocarriers,preferably all of a dose of adjuvant(s) is coupled to syntheticnanocarriers. In embodiments, at least a portion of a dose ofadjuvant(s) is not coupled to synthetic nanocarriers.

“Dosage form” means a pharmacologically and/or immunologically activematerial in a medium, carrier, vehicle, or device suitable foradministration to a subject. In embodiments, at least one inventivedosage form can comprise a dose of an adjuvant or multiple adjuvants. Inembodiments, more than one dosage form comprise a dose of adjuvant,preferably in such embodiments the more than one dosage forms areco-administered.

“Encapsulate” means to enclose within a synthetic nanocarrier,preferably enclose completely within a synthetic nanocarrier. Most orall of a substance that is encapsulated is not exposed to the localenvironment external to the synthetic nanocarrier. Encapsulation isdistinct from absorption, which places most or all of a substance on asurface of a synthetic nanocarrier, and leaves the substance exposed tothe local environment external to the synthetic nanocarrier.

“Generating” means causing an action, such as an antibody titer againstan antigen or systemic cytokine release, to occur, either directlyoneself or indirectly, such as, but not limited to, an unrelated thirdparty that takes an action through reliance on one's words or deeds.

An “infection” or “infectious disease” is any condition or diseasecaused by a microorganism, pathogen or other agent, such as a bacterium,fungus, prion or virus.

“Isolated nucleic acid” means a nucleic acid that is separated from itsnative environment and present in sufficient quantity to permit itsidentification or use. An isolated nucleic acid may be one that is (i)amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art. Any of the nucleic acids provided herein may be isolated. Insome embodiments, the antigens in the compositions provided herein arepresent in the form of an isolated nucleic acid, such as an isolatednucleic acid that encodes an antigenic peptide, polypeptide or protein.

“Isolated peptide, polypeptide or protein” means the polypeptide (orpeptide or protein) is separated from its native environment and presentin sufficient quantity to permit its identification or use. This means,for example, the polypeptide (or peptide or protein) may be (i)selectively produced by expression cloning or (ii) purified as bychromatography or electrophoresis. Isolated peptides, proteins orpolypeptides may be, but need not be, substantially pure. Because anisolated peptide, polypeptide or protein may be admixed with apharmaceutically acceptable carrier in a pharmaceutical preparation, thepolypeptide (or peptide or protein) may comprise only a small percentageby weight of the preparation. The polypeptide (or peptide or protein) isnonetheless isolated in that it has been separated from the substanceswith which it may be associated in living systems, i.e., isolated fromother proteins (or peptides or polypeptides). Any of the peptides,polypeptides or proteins provided herein may be isolated. In someembodiments, the antigens in the compositions provided herein are in theform of peptides, polypeptides or proteins.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheriodal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cuboidal synthetic nanocarrier, theminimum dimension of a synthetic nanocarrier would be the smallest ofits height, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is greater than 100 nm. In an embodiment, a maximum dimension ofat least 75%, preferably at least 80%, more preferably at least 90%, ofthe synthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample, is equal to or less than 5 μm.Preferably, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is greater than 110 nm, more preferably greater than 120 nm,more preferably greater than 130 nm, and more preferably still greaterthan 150 nm. Aspects ratios of the maximum and minimum dimensions ofinventive synthetic nanocarriers may vary depending on the embodiment.For instance, aspect ratios of the maximum to minimum dimensions of thesynthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from1:1 to 100,000:1, more preferably from 1:1 to 1000:1, still preferablyfrom 1:1 to 100:1, and yet more preferably from 1:1 to 10:1. Preferably,a maximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample isequal to or less than 3 μm, more preferably equal to or less than 2 μm,more preferably equal to or less than 1 μm, more preferably equal to orless than 800 nm, more preferably equal to or less than 600 nm, and morepreferably still equal to or less than 500 nm. In preferred embodiments,a maximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample, isequal to or greater than 100 nm, more preferably equal to or greaterthan 120, more preferably equal to or greater than 130 nm, morepreferably equal to or greater than 140 nm, and more preferably stillequal to or greater than 150 nm. Measurement of synthetic nanocarriersizes is obtained by suspending the synthetic nanocarriers in a liquid(usually aqueous) media and using dynamic light scattering (e.g. using aBrookhaven ZetaPALS instrument).

“Pharmaceutically acceptable carrier or excipient” means apharmacologically inactive material used together with the recitedsynthetic nanocarriers to formulate the inventive compositions.Pharmaceutically acceptable carriers or excipients comprise a variety ofmaterials known in the art, including but not limited to, saccharides(such as glucose, lactose and the like), preservatives such asantimicrobial agents, reconstitution aids, colorants, saline (such asphosphate buffered saline) and buffers. In some embodiments,pharmaceutically acceptable carriers or excipients comprise calciumcarbonate, calcium phosphate, various diluents, various sugars and typesof starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

“Subject” means animals, including warm blooded mammals such as humansand primates; avians; domestic household or farm animals such as cats,dogs, sheep, goats, cattle, horses and pigs; laboratory animals such asmice, rats and guinea pigs; fish; reptiles; zoo and wild animals; andthe like.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Albumin nanoparticles are generally includedas synthetic nanocarriers, however in certain embodiments the syntheticnanocarriers do not comprise albumin nanoparticles. In embodiments,inventive synthetic nanocarriers do not comprise chitosan.

A synthetic nanocarrier can be, but is not limited to, one or aplurality of lipid-based nanoparticles(e.g. liposomes) (also referred toherein as lipid nanoparticles, i.e., nanoparticles where the majority ofthe material that makes up their structure are lipids), polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles(i.e., particlesthat are primarily made up of viral structural proteins but that are notinfectious or have low infectivity), peptide or protein-based particles(also referred to herein as protein particles, i.e., particles where themajority of the material that makes up their structure are peptides orproteins) (such as albumin nanoparticles) and/or nanoparticles that aredeveloped using a combination of nanomaterials such as lipid-polymernanoparticles. Synthetic nanocarriers may be a variety of differentshapes, including but not limited to spheroidal, cuboidal, pyramidal,oblong, cylindrical, toroidal, and the like. Synthetic nanocarriersaccording to the invention comprise one or more surfaces, including butnot limited to internal surfaces (surfaces generally facing an interiorportion of the synthetic nanocarrier) and external surfaces (surfacesgenerally facing an external environment of the synthetic nanocarrier).Exemplary synthetic nanocarriers that can be adapted for use in thepractice of the present invention comprise: (1) the biodegradablenanoparticles disclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2)the polymeric nanoparticles of Published US Patent Application20060002852 to Saltzman et al., (4) the lithographically constructednanoparticles of Published US Patent Application 20090028910 to DeSimoneet al., (5) the disclosure of WO 2009/051837 to von Andrian et al., or(6) the nanoparticles disclosed in Published US Patent Application2008/0145441 to Penades et al.

Synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than 100 nm, do not comprise asurface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do notsubstantially activate complement. In a more preferred embodiment,synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not activate complement. In embodiments, synthetic nanocarriersmay possess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3,1:5, 1:7, or greater than 1:10.

“Systemic cytokine release” means the systemic release of one or moreparticular cytokines. In some embodiments, the systemic cytokine releaseis a particular systemic cytokine release profile. In some embodiments,the particular systemic cytokine release, preferably a particularsystemic cytokine release profile, is in a human. In embodiments, thecompositions and methods provided herein (where at least a portion of adose of adjuvant is coupled to nanocarriers result in a particularsystemic cytokine release profile in a subject). The term “separate” or“separately” is also used to mean adjuvant that is not coupled to anysynthetic nanocarriers. Additionally, “systemic cytokine releaseprofile” means a pattern of systemic cytokine release, wherein thepattern comprises cytokine levels measured for several differentsystemic cytokines. In some embodiments, the particular systemiccytokine release profile comprises the systemic release of TNF-α, IL-6and/or IL-12. In other embodiments, the particular systemic cytokinerelease profile comprises the systemic release of IFN-γ, IL12 and/orIL-18.

“T cell antigen” means any antigen that is recognized by and triggers animmune response in a T cell (e.g., an antigen that is specificallyrecognized by a T cell receptor on a T cell or an NKT cell viapresentation of the antigen or portion thereof bound to a Class I orClass II major histocompatability complex molecule (MHC), or bound to aCD1 complex.) In some embodiments, an antigen that is a T cell antigenis also a B cell antigen. In other embodiments, the T cell antigen isnot also a B cell antigen. T cell antigens generally are proteins,polypeptides or peptides. T cell antigens may be an antigen thatstimulates a CD8+ T cell response, a CD4+ T cell response, or both. Thenanocarriers, therefore, in some embodiments can effectively stimulateboth types of responses.

In some embodiments the T cell antigen is a ‘universal’ T cell antigen,or T cell memory antigen, (i.e., one to which a subject has apre-existing memory and that can be used to boost T cell help to anunrelated antigen, for example an unrelated B cell antigen). Universal Tcell antigens include tetanus toxoid, as well as one or more peptidesderived from tetanus toxoid, Epstein-Barr virus, or influenza virus.Universal T cell antigens also include a components of influenza virus,such as hemagglutinin, neuraminidase, or nuclear protein, or one or morepeptides derived therefrom. In some embodiments, the universal T cellantigen is not one that is presented in a complex with a MHC molecule.In some embodiments, the universal T cell antigen is not complexed witha MHC molecule for presentation to a T helper cell. Accordingly, in someembodiments, the universal T cell antigen is not a T helper cellantigen. However, in other embodiments, the universal T cell antigen isa T helper cell antigen.

In embodiments, a T-helper cell antigen may comprise one or morepeptides obtained or derived from tetanus toxoid, Epstein-Barr virus,influenza virus, respiratory syncytial virus, measles virus, mumpsvirus, rubella virus, cytomegalovirus, adenovirus, diphtheria toxoid, ora PADRE peptide (known from the work of Sette et al. U.S. Pat. No.7,202,351). In other embodiments, a T-helper cell antigen may compriseovalbumin or a peptide obtained or derived therefrom. Preferably, theovalbumin comprises the amino acid sequence as set forth in AccessionNo. AAB59956, NP_990483.1, AAA48998, or CAA2371. In other embodiments,the peptide obtained or derived from ovalbumin comprises the followingamino acid sequence:H-Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg-OH(SEQ ID NO: 1). In other embodiments, a T-helper cell antigen maycomprise one or more lipids, or glycolipids, including but not limitedto: α-galactosylceramide (α-GalCer), α-linked glycosphingolipids (fromSphingomonas spp.), galactosyl diacylglycerols (from Borreliaburgdorferi), lypophosphoglycan (from Leishmania donovani), andphosphatidylinositol tetramannoside (PIM4) (from Mycobacterium leprae).For additional lipids and/or glycolipids useful as T-helper cellantigen, see V. Cerundolo et al., “Harnessing invariant NKT cells invaccination strategies.” Nature Rev Immun, 9:28-38 (2009).

In embodiments, CD4+ T-cell antigens may be derivatives of a CD4+ T-cellantigen that is obtained from a source, such as a natural source. Insuch embodiments, CD4+ T-cell antigen sequences, such as those peptidesthat bind to MHC II, may have at least 70%, 80%, 90%, or 95% identity tothe antigen obtained from the source. In embodiments, the T cellantigen, preferably a universal T cell antigen or T-helper cell antigen,may be coupled to, or uncoupled from, a synthetic nanocarrier. In someembodiments, the universal T cell antigen or T-helper cell antigen isencapsulated in the synthetic nanocarriers of the inventivecompositions.

“Vaccine” means a composition of matter that improves the immuneresponse to a particular pathogen or disease. A vaccine typicallycontains factors that stimulate a subject's immune system to recognize aspecific antigen as foreign and eliminate it from the subject's body. Avaccine also establishes an immunologic ‘memory’ so the antigen will bequickly recognized and responded to if a person is re-challenged.Vaccines can be prophylactic (for example to prevent future infection byany pathogen), or therapeutic (for example a vaccine against a tumorspecific antigen for the treatment of cancer or against an antigenderived from an infectious agent for the treatment of an infection orinfectious disease). In embodiments, a vaccine may comprise dosage formsaccording to the invention. Preferably, in some embodiments, thevaccines comprise an adjuvant (or adjuvants) coupled to a syntheticnanocarrier.

In specific embodiments, the inventive compositions incorporateadjuvants that comprise agonists for toll-like receptors (TLRs) 7 & 8(“TLR 7/8 agonists”). Of utility are the TLR 7/8 agonist compoundsdisclosed in U.S. Pat. No. 6,696,076 to Tomai et al., including but notlimited to imidazoquinoline amines, imidazopyridine amines, 6,7-fusedcycloalkylimidazopyridine amines, and 1,2-bridged imidazoquinolineamines. Preferred adjuvants comprise imiquimod and R848.

In specific embodiments, the inventive compositons incorporate adjuvantsthat comprise a ligand for Toll-like receptor (TLR)-9, such asimmunostimulatory DNA molecules comprising CpGs, which induce type Iinterferon secretion, and stimulate T and B cell activation leading toincreased antibody production and cytotoxic T cell responses (Krieg etal., CpG motifs in bacterial DNA trigger direct B cell activation.Nature. 1995. 374:546-549; Chu et al. CpG oligodeoxynucleotides act asadjuvants that switch on T helper 1 (Th1) immunity. J. Exp. Med. 1997.186:1623-1631; Lipford et al. CpG-containing synthetic oligonucleotidespromote B and cytotoxic T cell responses to protein antigen: a new classof vaccine adjuvants. Eur. J. Immunol. 1997. 27:2340-2344; Roman et al.Immunostimulatory DNA sequences function as T helper-1-promotingadjuvants. Nat. Med. 1997. 3:849-854; Davis et al. CpG DNA is a potentenhancer of specific immunity in mice immunized with recombinanthepatitis B surface antigen. J. Immunol. 1998. 160:870-876; Lipford etal., Bacterial DNA as immune cell activator. Trends Microbiol. 1998.6:496-500. In embodiments, CpGs may comprise modifications intended toenhance stability, such as phosphorothioate linkages, or othermodifications, such as modified bases. See, for example, U.S. Pat. Nos.5,663,153, 6,194,388, 7,262,286, or 7,276,489. In certain embodiments,to stimulate immunity rather than tolerance, a composition providedherein incorporates an adjuvant that promotes DC maturation (needed forpriming of naive T cells) and the production of cytokines, such as typeI interferons, which promote antibody responses and anti-viral immunity.In some embodiments, the adjuvant comprises a TLR-4 agonist, such asbacterial lipopolysacharide (LPS), VSV-G, and/or HMGB-1. In someembodiments, adjuvants comprise cytokines, which are small proteins orbiological factors (in the range of 5 kD-20 kD) that are released bycells and have specific effects on cell-cell interaction, communicationand behavior of other cells. In some embodiments, adjuvants compriseproinflammatory stimuli released from necrotic cells (e.g., uratecrystals). In some embodiments, adjuvants comprise activated componentsof the complement cascade (e.g., CD21, CD35, etc.). In some embodiments,adjuvants comprise activated components of immune complexes. Theadjuvants also include those that comprise complement receptor agonists,such as a molecule that binds to CD21 or CD35. In some embodiments, thecomplement receptor agonist induces endogenous complement opsonizationof the nanocarrier. Adjuvants also include those that comprise cytokinereceptor agonists, such as a cytokine.

In some embodiments, the cytokine receptor agonist is a small molecule,antibody, fusion protein, or aptamer. In embodiments, adjuvants also maycomprise immunostimulatory RNA molecules, such as but not limited todsRNA or poly I:C (a TLR3 stimulant), and/or those disclosed in F. Heilet al., “Species-Specific Recognition of Single-Stranded RNA viaToll-like Receptor 7 and 8” Science 303(5663), 1526-1529 (2004); J.Vollmer et al., “Immune modulation by chemically modifiedribonucleosides and oligoribonucleotides” WO 2008033432 A2; A. Forsbachet al., “Immunostimulatory oligoribonucleotides containing specificsequence motif(s) and targeting the Toll-like receptor 8 pathway” WO2007062107 A2; E. Uhlmann et al., “Modified oligoribonucleotide analogswith enhanced immunostimulatory activity” U.S. Pat. Appl. Publ. US2006241076; G. Lipford et al., “Immunostimulatory viral RNAoligonucleotides and use for treating cancer and infections” WO2005097993 A2; G. Lipford et al., “Immunostimulatory G,U-containingoligoribonucleotides, compositions, and screening methods” WO 2003086280A2.

In some embodiments, the adjuvants comprise gel-type adjuvants (e.g.,aluminum hydroxide, aluminum phosphate, calcium phosphate, etc.),microbial adjuvants (e.g., immunomodulatory DNA sequences that includeCpG motifs; immunostimulatory RNA molecules; endotoxins such asmonophosphoryl lipid A; exotoxins such as cholera toxin, E. coli heatlabile toxin, and pertussis toxin; muramyl dipeptide, etc.);oil-emulsion and emulsifier-based adjuvants (e.g., Freund's Adjuvant,MF59 [Novartis], SAF, etc.); particulate adjuvants (e.g., liposomes,biodegradable microspheres, saponins, etc.); synthetic adjuvants (e.g.,nonionic block copolymers, muramyl peptide analogues, polyphosphazene,synthetic polynucleotides, etc.), and/or combinations thereof.

Inventive Compositions

A wide variety of synthetic nanocarriers can be used according to theinvention. In some embodiments, synthetic nanocarriers are spheres orspheroids. In some embodiments, synthetic nanocarriers are flat orplate-shaped. In some embodiments, synthetic nanocarriers are cubes orcuboidal. In some embodiments, synthetic nanocarriers are ovals orellipses. In some embodiments, synthetic nanocarriers are cylinders,cones, or pyramids.

In some embodiments, it is desirable to use a population of syntheticnanocarriers that is relatively uniform in terms of size, shape, and/orcomposition so that each synthetic nanocarrier has similar properties.For example, at least 80%, at least 90%, or at least 95% of thesynthetic nanocarriers, based on the total number of syntheticnanocarriers, may have a minimum dimension or maximum dimension thatfalls within 5%, 10%, or 20% of the average diameter or averagedimension of the synthetic nanocarriers. In some embodiments, apopulation of synthetic nanocarriers may be heterogeneous with respectto size, shape, and/or composition.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g. a polymeric core) and the shell is a secondlayer (e.g. a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more lipids. In some embodiments, a synthetic nanocarrier maycomprise a liposome. In some embodiments, a synthetic nanocarrier maycomprise a lipid bilayer. In some embodiments, a synthetic nanocarriermay comprise a lipid monolayer. In some embodiments, a syntheticnanocarrier may comprise a micelle. In some embodiments, a syntheticnanocarrier may comprise a core comprising a polymeric matrix surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In someembodiments, a synthetic nanocarrier may comprise a non-polymeric core(e.g., metal particle, quantum dot, ceramic particle, bone particle,viral particle, proteins, nucleic acids, carbohydrates, etc.) surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).

In some embodiments, synthetic nanocarriers can comprise one or morepolymers or polymeric matrices. In some embodiments, such a polymer orpolymeric matrix can be surrounded by a coating layer (e.g., liposome,lipid monolayer, micelle, etc.). In some embodiments, various elementsof the synthetic nanocarriers can be coupled with the polymer orpolymeric matrix.

In some embodiments, an element, such as an immunofeature surface,targeting moiety, antigen, adjuvant, and/or oligonucleotide can becovalently associated with a polymeric matrix. In some embodiments,covalent association is mediated by a linker. In some embodiments, anelement can be noncovalently associated with a polymeric matrix. Forexample, in some embodiments, an element can be encapsulated within,surrounded by, and/or dispersed throughout a polymeric matrix.Alternatively or additionally, an element can be associated with apolymeric matrix by hydrophobic interactions, charge interactions, vander Waals forces, etc.

A wide variety of polymers and methods for forming polymeric matricestherefrom are known conventionally. In general, a polymeric matrixcomprises one or more polymers. Polymers may be natural or unnatural(synthetic) polymers. Polymers may be homopolymers or copolymerscomprising two or more monomers. In terms of sequence, copolymers may berandom, block, or comprise a combination of random and block sequences.Typically, polymers in accordance with the present invention are organicpolymers.

Examples of polymers suitable for use in the present invention include,but are not limited to polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polypropylfumerates, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide,polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g.,poly(β-hydroxyalkanoate)), poly(orthoesters), polycyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polystyrenes, polyamines, polylysine,polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. § 177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a synthetic nanocarrier comprising a hydrophilic polymericmatrix generates a hydrophilic environment within the syntheticnanocarrier. In some embodiments, polymers can be hydrophobic. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polymericmatrix generates a hydrophobic environment within the syntheticnanocarrier. Selection of the hydrophilicity or hydrophobicity of thepolymer may have an impact on the nature of materials that areincorporated (e.g. coupled) within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with polyethylene glycol (PEG), with acarbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certainembodiments may be made using the general teachings of U.S. Pat. No.5,543,158 to Gref et al., or WO publication WO2009/051837 by Von Andrianet al.

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, poly(caprolactone),poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc.Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines. In embodiments, the inventive synthetic nanocarriers maynot comprise (or may exclude) cationic polymers.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al.,1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al.,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing certain suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that inventive synthetic nanocarriers may comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any ofthe foregoing and other polymers. Those skilled in the art willrecognize that the polymers listed herein represent an exemplary, notcomprehensive, list of polymers that can be of use in accordance withthe present invention.

In some embodiments, the synthetic nanocarriers comprise one or morepolymers. The polymeric synthetic nanocarriers, therefore, can alsoinclude those described in WO publication WO2009/051837 by Von Andrianet al., including, but not limited to those, with one or morehydrophilic components. Preferably, the one or more polymers comprise apolyester, such as a poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid), or polycaprolactone. More preferably, theone or more polymers comprise or further comprise a polyester coupled toa hydrophilic polymer, such as a polyether. In embodiments, thepolyether comprises polyethylene glycol. Still more preferably, the oneor more polymers comprise a polyester and a polyester coupled to ahydrophilic polymer, such as a polyether. In other embodiments, the oneor more polymers are coupled to one or more antigens and/or one or moreadjuvants. In embodiments, at least some of the polymers are coupled tothe antigen(s) and/or at least some of the polymers are coupled to theadjuvant(s). Preferably, when there are more than one type of polymer,one of the types of polymer is coupled to the antigen(s). Inembodiments, one of the other types of polymer is coupled to theadjuvant(s). For example, in embodiments, when the nanocarriers comprisea polyester and a polyester coupled to a hydrophilic polymer, such as apolyether, the polyester is coupled to the adjuvant, while the polyestercoupled to the hydrophilic polymer, such as a polyether, is coupled tothe antigen(s). In embodiments, where the nanocarriers comprise a Thelper cell antigen, the T helper cell antigen can be encapsulated inthe nanocarrier.

In some embodiments, synthetic nanocarriers may not comprise a polymericcomponent. In some embodiments, synthetic nanocarriers may comprisemetal particles, quantum dots, ceramic particles, etc. In someembodiments, a non-polymeric synthetic nanocarrier is an aggregate ofnon-polymeric components, such as an aggregate of metal atoms (e.g.,gold atoms).

In some embodiments, synthetic nanocarriers may optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanocarriers with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanocarriers in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate(Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85(Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; asorbitan fatty acid ester such as sorbitan trioleate; lecithin;lysolecithin; phosphatidylserine; phosphatidylinositol;sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity may be used in the production ofsynthetic nanocarriers to be used in accordance with the presentinvention.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more carbohydrates. Carbohydrates may be natural or synthetic. Acarbohydrate may be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is a polysaccharide, including but not limited to pullulan,cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose(HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran,cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon,amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,starch, chitin, heparin, inulin, konjac, glucommannan, pustulan,heparin, hyaluronic acid, curdlan, and xanthan. In embodiments, theinventive synthetic nanocarriers do not comprise (or specificallyexclude) carbohydrates, such as a polysaccharide. In certainembodiments, the carbohydrate may comprise a carbohydrate derivativesuch as a sugar alcohol, including but not limited to mannitol,sorbitol, xylitol, erythritol, maltitol, and lactitol.

Compositions according to the invention comprise inventive syntheticnanocarriers in combination with pharmaceutically acceptable excipients,such as preservatives, buffers, saline, or phosphate buffered saline.The compositions may be made using conventional pharmaceuticalmanufacturing and compounding techniques to arrive at useful dosageforms. In an embodiment, inventive synthetic nanocarriers are suspendedin sterile saline solution for injection together with a preservative.

In embodiments, when preparing synthetic nanocarriers as carriers foragents (e.g., antigen or adjuvant) for use in vaccines, methods forcoupling the agents to the synthetic nanocarriers may be useful. If theagent is a small molecule it may be of advantage to attach the agent toa polymer prior to the assembly of the synthetic nanocarriers. Inembodiments, it may also be an advantage to prepare the syntheticnanocarriers with surface groups that are used to couple the agent tothe synthetic nanocarrier through the use of these surface groups ratherthan attaching the agent to a polymer and then using this polymerconjugate in the construction of synthetic nanocarriers. A variety ofreactions can be used for the purpose of attaching agents to syntheticnanocarriers.

In certain embodiments, the coupling can be a covalent linker. Inembodiments, peptides according to the invention can be covalentlycoupled to the external surface via a 1,2,3-triazole linker formed bythe 1,3-dipolar cycloaddition reaction of azido groups on the surface ofthe nanocarrier with antigen or adjuvant containing an alkyne group orby the 1,3-dipolar cycloaddition reaction of alkynes on the surface ofthe nanocarrier with antigens or adjuvants containing an azido group.Such cycloaddition reactions are preferably performed in the presence ofa Cu(I) catalyst along with a suitable Cu(I)-ligand and a reducing agentto reduce Cu(II) compound to catalytic active Cu(I) compound. ThisCu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be referredas the click reaction.

Additionally, the covalent coupling may comprise a covalent linker thatcomprises an amide linker, a disulfide linker, a thioether linker, ahydrazone linker, a hydrazide linker, an imine or oxime linker, an ureaor thiourea linker, an amidine linker, an amine linker, and asulfonamide linker.

An amide linker is formed via an amide bond between an amine on onecomponent such as the antigen or adjuvant with the carboxylic acid groupof a second component such as the nanocarrier. The amide bond in thelinker can be made using any of the conventional amide bond formingreactions with suitably protected amino acids or antigens or adjuvantsand activated carboxylic acid such N-hydroxysuccinimide-activated ester.

A disulfide linker is made via the formation of a disulfide (S—S) bondbetween two sulfur atoms of the form, for instance, of R1-S—S—R2. Adisulfide bond can be formed by thiol exchange of an antigen or adjuvantcontaining thiol/mercaptan group(-SH) with another activated thiol groupon a polymer or nanocarrier or a nanocarrier containing thiol/mercaptangroups with a antigen or adjuvant containing activated thiol group.

A triazole linker, specifically a 1,2,3-triazole of the form

wherein R1 and R2 may be any chemical entities, is made by the1,3-dipolar cycloaddition reaction of an azide attached to a firstcomponent such as the nanocarrier with a terminal alkyne attached to asecond component such as the peptide. The 1,3-dipolar cycloadditionreaction is performed with or without a catalyst, preferably withCu(I)-catalyst, which links the two components through a 1,2,3-triazolefunction. This chemistry is described in detail by Sharpless et al.,Angew. Chem. Int. Ed. 41(14), 2596, (2002) and Meldal, et al, Chem.Rev., 2008, 108(8), 2952-3015 and is often referred to as a “click”reaction or CuAAC.

In embodiments, a polymer containing an azide or alkyne group, terminalto the polymer chain is prepared. This polymer is then used to prepare asynthetic nanocarrier in such a manner that a plurality of the alkyne orazide groups are positioned on the surface of that nanocarrier.Alternatively, the synthetic nanocarrier can be prepared by anotherroute, and subsequently functionalized with alkyne or azide groups. Theantigen or adjuvant is prepared with the presence of either an alkyne(if the polymer contains an azide) or an azide (if the polymer containsan alkyne) group. The antigen or adjuvant is then allowed to react withthe nanocarrier via the 1,3-dipolar cycloaddition reaction with orwithout a catalyst which covalently couples the antigen or adjuvant tothe particle through the 1,4-disubstituted 1,2,3-triazole linker.

A thioether linker is made by the formation of a sulfur-carbon(thioether) bond in the form, for instance, of R1-S—R2. Thioether can bemade by either alkylation of a thiol/mercaptan (—SH) group on onecomponent such as the antigen or adjuvant with an alkylating group suchas halide or epoxide on a second component such as the nanocarrier.Thioether linkers can also be formed by Michael addition of athiol/mercaptan group on one component such as a antigen or adjuvant toan electron-deficient alkene group on a second component such as apolymer containing a maleimide group or vinyl sulfone group as theMichael acceptor. In another way, thioether linkers can be prepared bythe radical thiol-ene reaction of a thiol/mercaptan group on onecomponent such as a antigen or adjuvant with an alkene group on a secondcomponent such as a polymer or nanocarrier.

A hydrazone linker is made by the reaction of a hydrazide group on onecomponent such as the antigen or adjuvant with an aldehyde/ketonechemistry group on the second component such as the nanocarrier.

A hydrazide linker is formed by the reaction of a hydrazine group on onecomponent such as the antigen or adjuvant with a carboxylic acid groupon the second component such as the nanocarrier. Such reaction isgenerally performed using chemistry similar to the formation of amidebond where the carboxylic acid is activated with an activating reagent.

An imine or oxime linker is formed by the reaction of an amine orN-alkoxyamine (or aminooxy) group on one component such as the antigenor adjuvant with an aldehyde or ketone group on the second componentsuch as the nanocarrier.

An urea or thiourea linker is prepared by the reaction of an amine groupon one component such as the antigen or adjuvant with an isocyanate orthioisocyanate group on the second component such as the nanocarrier.

An amidine linker is prepared by the reaction of an amine group on onecomponent such as the antigen or adjuvant with an imidoester group onthe second component such as the nanocarrier.

An amine linker is made by the alkylation reaction of an amine group onone component such as the antigen or adjuvant with an alkylating groupsuch as halide, epoxide, or sulfonate ester group on the secondcomponent such as the nanocarrier. Alternatively, an amine linker canalso be made by reductive amination of an amine group on one componentsuch as the antigen or adjuvant with an aldehyde or ketone group on thesecond component such as the nanocarrier with a suitable reducingreagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.

A sulfonamide linker is made by the reaction of an amine group on onecomponent such as the antigen or adjuvant with a sulfonyl halide (suchas sulfonyl chloride) group on the second component such as thenanocarrier.

A sulfone linker is made by Michael addition of a nucleophile to a vinylsulfone. Either the vinyl sulfone or the nucleophile may be on thesurface of the nanoparticle or attached to the antigen or adjuvant.

The antigen or adjuvant can also be conjugated to the nanocarrier vianon-covalent conjugation methods. For examples, a negative chargedantigen or adjuvant can be conjugated to a positive charged nanocarrierthrough electrostatic adsorption. An antigen or adjuvant containing ametal ligand can also be conjugated to a nanocarrier containing a metalcomplex via a metal-ligand complex.

In embodiments, the antigen or adjuvant can be attached to a polymer,for example polylactic acid-block-polyethylene glycol, prior to theassembly of the synthetic nanocarrier or the synthetic nanocarrier canbe formed with reactive or activatible groups on its surface. In thelatter case, the antigen or adjuvant is prepared with a group which iscompatible with the attachment chemistry that is presented by thesynthetic nanocarriers' surface. In other embodiments, agents, such as apeptide antigen, can be attached to VLPs or liposomes using a suitablelinker. A linker is a compound or reagent that capable of coupling twomolecules together. In an embodiment, the linker can be ahomobifuntional or heterobifunctional reagent as described in Hermanson2008. For example, an VLP or liposome synthetic nanocarrier containing acarboxylic group on the surface can be treated with a homobifunctionallinker, adipic dihydrazide (ADH), in the presence of EDC to form thecorresponding synthetic nanocarrier with the ADH linker. The resultingADH linked synthetic nanocarrier is then conjugated with an agentcontaining an acid group via the other end of the ADH linker on the NCto produce the corresponding VLP or liposome peptide conjugate.

For detailed descriptions of available conjugation methods, seeHermanson G T “Bioconjugate Techniques”, 2nd Edition Published byAcademic Press, Inc., 2008. In addition to covalent attachment theantigen or adjuvant can be coupled by adsorbtion to a pre-formedsynthetic nanocarrier or it can be coupled by encapsulation during theformation of the synthetic nanocarrier.

Methods of Making and Using the Inventive Methods and RelatedCompositions

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods as nanoprecipitation, flow focusing using fluidic channels,spray drying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanomaterials have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843). Additional methods havebeen described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755, U.S. Pat. Nos. 5,578,325 and6,007,845; P. Paolicelli et al., “Surface-modified PLGA-basedNanoparticles that can Efficiently Associate and Deliver Virus-likeParticles” Nanomedicine. 5(6):843-853 (2010)).

Various materials may be encapsulated into synthetic nanocarriers asdesirable using a variety of methods including but not limited to C.Astete et al., “Synthesis and characterization of PLGA nanoparticles” J.Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K.Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide)Nanoparticles: Preparation, Properties and Possible Applications in DrugDelivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al.,“Nanoencapsulation I. Methods for preparation of drug-loaded polymericnanoparticles” Nanomedicine 2:8-21 (2006); P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010). Other methods suitable for encapsulating materials, such asoligonucleotides, into synthetic nanocarriers may be used, includingwithout limitation methods disclosed in U.S. Pat. No. 6,632,671 to UngerOct. 14, 2003.

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe coupled to the synthetic nanocarriers and/or the composition of thepolymer matrix.

If particles prepared by any of the above methods have a size rangeoutside of the desired range, particles can be sized, for example, usinga sieve.

Elements of the inventive synthetic nanocarriers—such as targetingmoieties, polymeric matrices, antigens, adjuvants and the like—may becoupled to the synthetic nanocarrier, e.g., by one or more covalentbonds, or may be coupled by means of one or more linkers. Additionalmethods of functionalizing synthetic nanocarriers may be adapted fromPublished US Patent Application 2006/0002852 to Saltzman et al.,Published US Patent Application 2009/0028910 to DeSimone et al., orPublished International Patent Application WO/2008/127532 A1 to Murthyet al.

Alternatively or additionally, synthetic nanocarriers can be coupled toan element, such as immunofeature surfaces, targeting moieties,adjuvants, various antigens, etc. directly or indirectly vianon-covalent interactions. In non-covalent embodiments, the non-covalentcoupling is mediated by non-covalent interactions including but notlimited to charge interactions, affinity interactions, metalcoordination, physical adsorption, host-guest interactions, hydrophobicinteractions, TT stacking interactions, hydrogen bonding interactions,van der Waals interactions, magnetic interactions, electrostaticinteractions, dipole-dipole interactions, and/or combinations thereof.Such couplings may be arranged to be on an external surface or aninternal surface of an inventive synthetic nanocarrier. In embodiments,encapsulation and/or absorption is a form of coupling.

In embodiments, the inventive synthetic nanocarriers can be combinedwith other adjuvants by admixing in the same vehicle or delivery system.Such adjuvants may include, but are not limited to mineral salts, suchas alum, alum combined with monphosphoryl lipid (MPL) A ofEnterobacteria, such as Escherihia coli, Salmonella minnesota,Salmonella typhimurium, or Shigella flexneri or specifically with MPL®(AS04), AS15, MPL A of above-mentioned bacteria separately, saponins,such as QS-21,Quil-A, ISCOMs, ISCOMATRIX™, emulsions such as MF59™,Montanide® ISA 51 and ISA 720, AS02 (QS21+squalene+MPL®), liposomes andliposomal formulations such as ASO1, synthesized or specificallyprepared microparticles and microcarriers such as bacteria-derived outermembrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis andothers, or chitosan particles, depot-forming agents, such as Pluronic®block co-polymers, specifically modified or prepared peptides, such asmuramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529,or proteins, such as bacterial toxoids or toxin fragments. Additionaluseful adjuvants may be found in WO 2002/032450; U.S. Pat. No. 7,357,936“Adjuvant Systems and Vaccines”; U.S. Pat. No. 7,147,862 “Vaccinecomposition containing adjuvants”; U.S. Pat. No. 6,544,518 “Vaccines”;U.S. Pat. No. 5,750,110 “Vaccine composition containing adjuvants.” Thedoses of such other adjuvants can be determined using conventional doseranging studies. In embodiments, adjuvant that is not coupled to therecited synthetic nanocarriers, if any, may be the same or differentfrom adjuvant that is coupled to the synthetic nanocarriers

In embodiments, any adjuvant coupled to the inventive syntheticnanocarriers can be different, similar or identical to those not coupledto a nanocarrier (with or without antigen, utilizing or not utilizinganother delivery vehicle). The adjuvants (coupled and not coupled) canbe administered separately at a different time-point and/or at adifferent body location and/or by a different immunization route or withanother adjuvant-carrying synthetic nanocarrier (with or withoutantigen) administered separately at a different time-point and/or at adifferent body location and/or by a different immunization route.

Populations of synthetic nanocarriers may be combined to formpharmaceutical dosage forms according to the present invention usingtraditional pharmaceutical mixing methods. These include liquid-liquidmixing in which two or more suspensions, each containing one or moresubset of nanocarriers, are directly combined or are brought togethervia one or more vessels containing diluent. As synthetic nanocarriersmay also be produced or stored in a powder form, dry powder-powdermixing could be performed as could the re-suspension of two or morepowders in a common media. Depending on the properties of thenanocarriers and their interaction potentials, there may be advantagesconferred to one or another route of mixing.

Typical inventive compositions that can be used in the inventive methodscomprise synthetic nanocarriers may comprise inorganic or organicbuffers (e.g., sodium or potassium salts of phosphate, carbonate,acetate, or citrate) and pH adjustment agents (e.g., hydrochloric acid,sodium or potassium hydroxide, salts of citrate or acetate, amino acidsand their salts) antioxidants (e.g., ascorbic acid, alpha-tocopherol),surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10nonyl phenol, sodium desoxycholate), solution and/or cryo/lyostabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmoticadjustment agents (e.g., salts or sugars), antibacterial agents (e.g.,benzoic acid, phenol, gentamicin), antifoaming agents (e.g.,polydimethylsilozone), preservatives (e.g., thimerosal,2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity-adjustmentagents (e.g., polyvinylpyrrolidone, poloxamer 488,carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethyleneglycol, ethanol).

Compositions that can be used in the methods according to the inventioncomprise inventive synthetic nanocarriers in combination withpharmaceutically acceptable excipients. The compositions may be madeusing conventional pharmaceutical manufacturing and compoundingtechniques to arrive at useful dosage forms. Techniques suitable for usein practicing the present invention may be found in Handbook ofIndustrial Mixing: Science and Practice, Edited by Edward L. Paul,Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons,Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed.Edited by M. E. Auten, 2001, Churchill Livingstone. In an embodiment,inventive synthetic nanocarriers are suspended in sterile salinesolution for injection together with a preservative.

It is to be understood that the compositions that can be used in themethods of the invention can be made in any suitable manner, and theinvention is in no way limited to the use of compositions that can beproduced using the methods described herein. Selection of an appropriatemethod may require attention to the properties of the particularmoieties being associated.

In some embodiments, inventive synthetic nanocarriers are manufacturedunder sterile conditions or are terminally sterilized. This can ensurethat resulting composition are sterile and non-infectious, thusimproving safety when compared to non-sterile compositions. Thisprovides a valuable safety measure, especially when subjects receivingsynthetic nanocarriers have immune defects, are suffering frominfection, and/or are susceptible to infection. In some embodiments,inventive synthetic nanocarriers may be lyophilized and stored insuspension or as lyophilized powder depending on the formulationstrategy for extended periods without losing activity.

The compositions that can be used in the inventive methods may beadministered by a variety of routes of administration, including but notlimited to subcutaneous, intramuscular, intradermal, oral, intranasal,transmucosal, sublingual, rectal, ophthalmic, transdermal,transcutaneous or by a combination of these routes.

Doses of dosage forms contain varying amounts of populations ofsynthetic nanocarriers and/or varying amounts of adjuvants and/orantigens, according to the invention. The amount of syntheticnanocarriers and/or adjuvants and/or antigens present in the inventivedosage forms can be varied according to the nature of the adjuvantsand/or antigens, the therapeutic benefit to be accomplished, and othersuch parameters. In some embodiments, the doses of the dosage forms aresub-therapeutic or toxicity-reduced doses. In other embodiments, thedoses are amounts effective to generate one or more immune responses asprovided herein. In some embodiments, the immune response(s) is anantibody response or generation of an antibody titer and/or systemiccytokine release. In embodiments, dose ranging studies can be conductedto establish optimal therapeutic amount of the population of syntheticnanocarriers and/or the amount of adjuvants and/or antigens to bepresent in the dosage form. In embodiments, the synthetic nanocarriersand/or the adjuvants and/or antigens are present in the dosage form inan amount effective to generate an immune response as provided hereinupon administration to a subject. In some embodiments, the subject is ahuman. It may be possible to determine amounts of the adjuvants and/orantigens effective to generate an immune response as provided hereinusing conventional dose ranging studies and techniques in subjects.Inventive dosage forms may be administered at a variety of frequencies.In a preferred embodiment, at least one administration of the dosageform is sufficient to generate a pharmacologically relevant response. Inmore preferred embodiment, at least two administrations, at least threeadministrations, or at least four administrations, of the dosage formare utilized to ensure a pharmacologically relevant response.

The compositions and methods described herein can be used to induce,enhance, stimulate, modulate, direct or redirect an immune response. Thecompositions and methods described herein can be used in the diagnosis,prophylaxis and/or treatment of conditions such as cancers, infectiousdiseases, metabolic diseases, degenerative diseases, autoimmunediseases, inflammatory diseases, immunological diseases, or otherdisorders and/or conditions. The compositions and methods describedherein can also be used for the prophylaxis or treatment of anaddiction, such as an addiction to nicotine or a narcotic. Thecompositions and methods described herein can also be used for theprophylaxis and/or treatment of a condition resulting from the exposureto a toxin, hazardous substance, environmental toxin, or other harmfulagent.

In embodiments, the compositions and methods provided can be used tosystemically induce cytokines, such as TNF-α, IL-6 and/or IL-12, orIFN-γ, IL-12 and/or IL-18. In other embodiments, the compositions andmethods provided can be used to induce an antibody response or togenerate an antibody titer. The immune responses as provided herein canbe specific to an antigen, such as any of the antigens provided herein,preferably to one or more antigens in an inventive composition or thatis administered according to an inventive method provided herein.

The compositions and methods provided herein can be used in a variety ofsubjects. The subjects provided herein can have or be at risk of havingcancer. Cancers include, but are not limited to, breast cancer; biliarytract cancer; bladder cancer; brain cancer including glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; hematologicalneoplasms including acute lymphocytic and myelogenous leukemia, e.g., BCell CLL; T-cell acute lymphoblastic leukemia/lymphoma; hairy cellleukemia; chronic myelogenous leukemia, multiple myeloma;AIDS-associated leukemias and adult T-cell leukemia/lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer; lymphomas including Hodgkin's disease andlymphocytic lymphomas; neuroblastomas; oral cancer including squamouscell carcinoma; ovarian cancer including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells; pancreaticcancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, andosteosarcoma; skin cancer including melanoma, Merkel cell carcinoma,Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer;testicular cancer including germinal tumors such as seminoma,non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germcell tumors; thyroid cancer including thyroid adenocarcinoma andmedullar carcinoma; and renal cancer including adenocarcinoma and Wilmstumor.

The subjects provided herein can have or be at risk of having aninfection or infectious disease. Infections or infectious diseasesinclude, but are not limited to, viral infectious diseases, such asAIDS, Chickenpox (Varicella), Common cold, Cytomegalovirus Infection,Colorado tick fever, Dengue fever, Ebola hemorrhagic fever, Hand, footand mouth disease, Hepatitis, Herpes simplex, Herpes zoster, HPV,Influenza (Flu), Lassa fever, Measles, Marburg hemorrhagic fever,Infectious mononucleosis, Mumps, Norovirus, Poliomyelitis, Progressivemultifocal leukencephalopathy, Rabies, Rubella, SARS, Smallpox(Variola), Viral encephalitis, Viral gastroenteritis, Viral meningitis,Viral pneumonia, West Nile disease and Yellow fever; bacterialinfectious diseases, such as Anthrax, Bacterial Meningitis, Botulism,Brucellosis, Campylobacteriosis, Cat Scratch Disease, Cholera,Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo, Legionellosis, Leprosy(Hansen's Disease), Leptospirosis, Listeriosis, Lyme disease,Melioidosis, Rheumatic Fever, MRSA infection, Nocardiosis, Pertussis(Whooping Cough), Plague, Pneumococcal pneumonia, Psittacosis, Q fever,Rocky Mountain Spotted Fever (RMSF), Salmonellosis, Scarlet Fever,Shigellosis, Syphilis, Tetanus, Trachoma, Tuberculosis, Tularemia,Typhoid Fever, Typhus and Urinary Tract Infections; parasitic infectiousdiseases, such as African trypanosomiasis, Amebiasis, Ascariasis,Babesiosis, Chagas Disease, Clonorchiasis, Cryptosporidiosis,Cysticercosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis,Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Free-livingamebic infection, Giardiasis, Gnathostomiasis, Hymenolepiasis,Isosporiasis, Kala-azar, Leishmaniasis, Malaria, Metagonimiasis,Myiasis, Onchocerciasis, Pediculosis, Pinworm Infection, Scabies,Schistosomiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinellosis,Trichinosis, Trichuriasis, Trichomoniasis and Trypanosomiasis; fungalinfectious disease, such as Aspergillosis, Blastomycosis, Candidiasis,Coccidioidomycosis, Cryptococcosis, Histoplasmosis, Tinea pedis(Athlete's Foot) and Tinea cruris; prion infectious diseases, such asAlpers' disease, Fatal Familial Insomnia, Gerstmann-Sträussler-Scheinkersyndrome, Kuru and Variant Creutzfeldt-Jakob disease.

Subjects provided here also include those that have or are at risk ofhaving an atopic condition, such as but not limited to allergy, allergicasthma, or atopic dermatitis; asthma; chronic obstructive pulmonarydisease (COPD, e.g. emphysema or chronic bronchitis); and chronicinfections due to chronic infectious agents such as chronicLeishmaniasis, candidiasis or schistosomiasis and infections caused byplasmodia, toxoplasma gondii, mycobacteria, HIV, HBV, HCV EBV or CMV, orany one of the above, or any subset of the above.

EXAMPLES Example 1 Synthetic Nanocarriers with Covalently CoupledAdjuvant (Prophetic)

Resiquimod (aka R848) is synthesized according to the synthesis providedin Example 99 of U.S. Pat. No. 5,389,640 to Gerster et al. PLA-R848conjugate is prepared. PLA-PEG-nicotine conjugate is prepared. PLA isprepared by a ring opening polymerization using D,L-lactide(MW=approximately 15 KD-18 KD). The PLA structure is confirmed by NMR.The polyvinyl alcohol (Mw=11 KD-31 KD, 85% hydrolyzed) is purchased fromVWR scientific.

These are used to prepare the following solutions:

1. PLA-R848 conjugate @ 100 mg/mL in methylene chloride

2. PLA-PEG-nicotine in methylene chloride @ 100 mg/mL

3. PLA in methylene chloride @ 100 mg/mL

4. Polyvinyl alcohol in water @50 mg/mL.

Solution #1 (0.25 to 0.75 mL), solution #2 (0.25 mL) and solution #3(0.25 to 0.5 mL) are combined in a small vial with distilled water (0.5mL), and the mixture is sonicated at 50% amplitude for 40 seconds usinga Branson Digital Sonifier 250. To this emulsion is added solution #4(2.0 mL) and sonication at 35% amplitude for 40 seconds using theBranson Digital Sonifier 250 forms the second emulsion. This is added toa beaker containing phosphate buffer solution (30 mL), and this mixtureis stirred at room temperature for 2 hours to form the nanocarriers. Towash the nanocarriers, a portion of the nanocarrier dispersion (7.0 mL)is transferred to a centrifuge tube and spun at 5,300 g for one hour,supernatant is removed, and the pellet is re-suspended in 7.0 mL ofphosphate buffered saline. The centrifuge procedure is repeated and thepellet is re-suspended in 2.2 mL of phosphate buffered saline for afinal nanocarrier dispersion of about 10 mg/mL.

Example 2 Synthetic Nanocarriers with Non-Covalently Coupled Adjuvant(Prophetic)

Charged nanocarriers are made as follows:

1. PLA-PEG-OMe in methylene chloride @ 100 mg/mL

2. PLA in methylene chloride @ 100 mg/mL

3. Cetyl trimethylammonium bromide (CTAB) in water at 5 mg/mL

Solution #1 (0.25 to 0.75 mL), solution #2 (0.25 mL) and distilled water(0.5 mL) are combined in a small vial and the mixture is sonicated at50% amplitude for 40 seconds using a Branson Digital Sonifier 250. Tothis emulsion is added solution #3 (2.0 mL) and sonication at 35%amplitude for 40 seconds using the Branson Digital Sonifier 250 formsthe second emulsion. This is added to a beaker containing phosphatebuffer solution (30 mL), and this mixture is stirred at room temperaturefor 2 hours to form the nanocarriers. To wash the nanocarriers a portionof the nanocarrier dispersion (7.0 mL) is transferred to a centrifugetube and spun at 5,300 g for one hour, supernatant is removed, and thepellet is re-suspended in 7.0 mL of phosphate buffered saline. Thecentrifuge procedure is repeated and the pellet is re-suspended in 2.2mL of DI water for a final nanocarrier dispersion of about 10 mg/mL. Toadsorb an antigen, in this case CpG DNA, to the nanocarriers, 1.0 mL ofthe charged nanocarriers in DI water at 10 mg/mL are cooled on ice. Tothis cooled suspension is added 10 μg of CpG DNA ODN 1826, and thismixture is incubated at 4° C. for 4 hours. The nanocarriers are thenisolated and washed as described above.

Example 3 Composition with Synthetic Nanocarriers and Uncoupled Antigen(Prophetic)

The polyvinyl alcohol (Mw=11 KD-31 KD, 87-89% partially hydrolyzed) ispurchased from J T Baker. Ovalbumin peptide 323-339 is obtained fromBachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Part#4065609). PLGA-R848 (or PLA-R848) and PLA-PEG-Antigen or PLA-PEG-Linkeror PLA-PEG-OMe conjugates are synthesized and purified.

The above materials are used to prepare the following solutions:

1. PLA-R848 or PLGA-R848 conjugate in methylene chloride @ 100 mg/mL

2. PLA-PEG-OMe in methylene chloride @ 100 mg/mL

3. PLA or PLGA in methylene chloride @ 100 mg/mL

4. Polyvinyl alcohol in 100 mM pH 8 phosphate buffer @50 mg/mL

Solution #1 (0.1 to 0.9 mL) and solution #2 (0.01 to 0.50 mL) arecombined, optionally also including solution #3 (0.1 to 0.89 mL),andthen distilled water (0.50 mL) is added in a small vessel and themixture is sonicated at 50% amplitude for 40 seconds using a BransonDigital Sonifier 250. To this emulsion is added solution #4 (2.0-3.0 mL)and sonication at 30% amplitude for 40 seconds using the Branson DigitalSonifier 250 forms the second emulsion. This is added to a stirringbeaker containing a 70 mM pH 8 phosphate buffer solution (30 mL), andthis mixture is stirred at room temperature for 2 hours to form thenanocarriers. To wash the nanocarriers, a portion of the nanocarrierdispersion (25 to 32 mL) is transferred to a 50 mL centrifuge tube andspun at 9500 rpm (13,800 g) for one hour at 4° C., supernatant isremoved, and the pellet is re-suspended in 25 to 32 mL of phosphatebuffered saline. The centrifugation procedure is repeated and the pelletis re-suspended in phosphate buffered saline to achieve a final nominalnanocarrier concentration of 10 mg/mL.

The nanocarriers are combined with the necessary amount of sterilesaline solution to reach the final concentration in a sterile vehicle,and then administered to a subject by subcutaneous or intramuscularinjection using a conventional slip-tip or Luer-lock syringe.

Example 4 Administration of Synthetic Nanocarriers andNon-Coadministered Antigen (Prophetic)

The synthetic nanocarriers of Example 3 are formulated into a sterilesaline vehicle, and then administered to a subject by subcutaneous orintramuscular injection using a conventional slip-tip or Luer-locksyringe. The subject is exposed to environmental antigen (e.g. pollen,animal antigens, etc.) that is not coadministered with the syntheticnanocarriers. Any altered immune response to the non-coadministeredantigen that is due to the administration of the synthetic nanocarriersis noted.

Example 5 Synthetic Nanocarriers with Covalently Coupled Adjuvant

Virus-like particles (VLP's) have received attention as nanocarriers foruse in vaccines and for drug delivery. These virus-like particles canalso be used to deliver covalently attached adjuvants. Virus-likeparticles can be made by a variety of methods, for example, as describedin Biotechnology and Bioengineering 100(1), 28, (2008). Covalentattachment can be accomplished as follows.

A suspension of virus-like particles in PBS (1.0 mL, 300 μg/mL) iscooled on ice. To this is added the R-848 conjugate (50 mg, describedbelow) in PBS (0.5 mL). EDC hydrochloride (50 mg) is added and themixture is gently stirred overnight at ice temperature. The resultingVLP conjugate is freed from excess R848 conjugate by dialysis.

The R848 conjugate is made as follows. R848 (5.0 gm, 1.59×10⁻² moles)and diglycolic anhydride (3.7 gm, 3.18×10⁻² moles) are combined indimethylacetamide (10 mL). This solution is heated at 120° C. for 2hours. After cooling slightly, 2-propanol (25 mL) is added ,and theresulting solution is stirred on ice for 1 hour. The imide separates asa white solid which is isolated by filtration, washed with 2-propanoland dried. The yield of the R848 imide is 6.45 gm (98%).

The R848 imide (412 mg, 1.0×10⁻³ moles) and 6-hydroxycaproic acid (132mg, 1.0×10⁻³ moles) are stirred in methylene chloride (5 mL). To thissuspension is added 1,5,7-triazabicyclo[4,4,0]dec-5-ene (TBD, 278 mg,2×10⁻³ moles) after which the suspension is stirred overnight at roomtemperature. The resulting clear solution is diluted with methylenechloride (25 mL), and this solution is washed with 5% citric acidsolution (2×25 mL). After drying over magnesium sulfate the solution isfiltered and evaporated under vacuum to provide the R848 conjugate usedin the VLP-antigen synthesis. The expected R848 conjugate structure isas follows:

Example 6 Coupling of Nanocarrier to R848 Adjuvant Abolishes SystemicProduction of Inflammatory Cytokines

Groups of mice were injected subcutaneously into hind limbs with 100 μgof nanocarriers (NC) coupled, non-coupled or admixed with small moleculenucleoside analogue and known TLR7/8 agonist and adjuvant R848. R848amount in nanocarrier was 2-3% resulting in 2-3 μg of coupled R848 perinjection; amount of free R848 used was 20 μg per injection. Mouse serumwas taken by terminal bleed and systemic cytokine production in serumwas measured at different time-points by ELISA (BD Biosciences). As seenin FIGS. 1A-1C, strong systemic production of major pro-inflammatorycytokines TNF-α, IL-6 and IL-12 was observed when admixed R848 (NC+R848)was used, while no expression of TNF-α, IL-6 and IL-12 was detected whentwo separate preparations of NC coupled with R848 (NC-R848-1 andNC-R848-2) were used. The difference in peak cytokine expression levelswas >100-fold for TNF-α and IL-6, and >50-fold for IL-12. NC not coupledto R848 (labeled as NC only) did not induce any systemic cytokines whenused without admixed R848.

Example 7 Coupling of Nanocarrier to R848 Adjuvant Does Not InhibitSystemic Production of Immune Cytokine IFN-γ

While early proinflammatory cytokines are associated with side effectsduring immunization, the production of other cytokines, such as immuneIFN-γ is known to be important for induction of effective immuneresponse. Therefore, an experiment was performed identically to that ofExample 6. Systemic production of immune cytokine IFN-γ (as measured inmouse serum by ELISA, BD Biosciences), which is instrumental for Th1immune response, was seen to reach the same level irrespectively ofwhether NC-R848 (containing 2 μg of R848) or NC with admixed R848 (20μg) was used (FIG. 2). Furthermore, the production of IFN-γ by NP-R848was distributed over a wider time window.

Example 8 The Production of Systemic IL-12 by Adjuvants R848 and CpG isAbolished by Their Coupling to Nanocarriers

The effect on systemic cytokine induction by coupling of a TLR agonistto a nanocarrier was demonstrated to not be specific to a particular TLRagonist. In this experiment groups of two mice were inoculated by freeTLR agonists R848 or CpG 1826 (20 μg each) and by the same moleculescoupled to nanocarriers, NC-R848 (100 μg of NC prep, containing a totalof 3 μg of R848) or NC-CpG (100 μg of NC prep, containing a total of 5μg of CpG 1826), and serum IL-12 measured at times indicated in pooledmouse sera (ELISA, BD Biosciences). As seen in FIG. 3, peak levels ofsystemic IL-12 were 30-fold higher by free R848 than by NC-R848 and20-fold higher by free CpG 1826 than by NC-CpG).

Example 9 Local Induction of Immune Cytokines IFN-γ, IL-12 and IL-1β isStrongly Augmented by Adjuvant Coupling to Nanocarriers, While Adjuvantis Spared

While systemic induction of pro-inflammatory cytokines is associatedwith adverse effects of vaccination, local induction of immunecytokines, such as IFN-γ or IL-1β, is viewed as mostly beneficial forthe induction of specific and localized immune response. In theexperiment shown in FIG. 4, mice were injected subcutaneously at thehind limb by free (20 μg) or NC-coupled R848 and CpG adjuvants (adjuvantcontent 2.5-4 μg), draining (popliteal) lymph nodes (LN) removed attimes indicated, incubated overnight in a standard cell culture mediumand cytokine production in cell supernatants measured by ELISA asdescribed above. Much stronger local induction of Th1 cytokines IFN-γ(50-100-fold, FIG. 4A) and IL-12 (17-fold, FIG. 4B) andinflammasome-related cytokine IL-1β (6-fold, FIG. 4C) was observed whenNC-R848 was used compared to free R848 (notably, the amounts of R848present in NC-R848 were 5-10 times less than of free R848). Similarly,NC-CpG was a much stronger inducer of local immune cytokines than freeCpG (known to be extremely potent in this regard). Local production ofIFN-γ was 7-15 times higher at peak levels (FIG. 4A), production ofIL-12 was 4 times higher (FIG. 4B), and production of IL-1β was 2 timeshigher (FIG. 4C). The amount of CpG 1826 present in NC-CpG was 4-5 timesless than of free CpG 1826.

Example 10 Local Lymph Node (LN) Stimulation and Induction of ImmuneCell Proliferation by NC-Coupled R848 Adjuvant, but not by Free R848

Draining lymph node swelling (lymphadenopathy) is a hallmark of localimmune activation. It results from infiltration of LN infiltration bydifferent cells instrumental for innate and adaptive immune response.Mice were subcutaneously inoculated with NC-R848, NC only or withNC-R848 in hind limbs as described above. Popliteal LNs were removed attimes indicated (FIG. 5), and the number of total cells as well asseparate immune cell population counted. Hemocytometer was used fortotal cell counts, and then cell populations were differentially stainedby surface cell markers and percentage of positive for each populationdetermined using FACS. DC: dendritic cells, mDC: myeloid DC, pDC:plasmacytoid DC, Mph: macrophages, Gr: granulocytes, B: B cells, T: Tcells, NK: natural killer cells. The following markers were used forstaining: CD11c⁺ (DC); CD11c⁺B220⁻ (mDC); CD11c⁺B220⁺ (pDC);F4/80+/Gr1⁻(Mph); F4/80⁻/Gr1+ (Gr); B220⁺CD11c⁻ (B cells); CD3⁺ (Tcells); CD3⁻/CD49b⁺ (NK cells). Major increase in total cell number indraining LN was seen after injection with NC-R848 with DC, granulocytes,B-cells and NK cells showing the most pronounced effect (FIG. 5).

Example 11 Antibody Response Higher for Nanocarrier with ConjugatedAdjuvant versus Admixed Adjuvant

Materials for Nic,R848,OP-II Nanocarrier Formulations

Ovalbumin peptide 323-339 amide TFA salt, was purchased from BachemAmericas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Part#4064565.) PLA with an inherent viscosity of 0.19 dL/g was purchasedfrom Boehringer Ingelheim (Ingelheim Germany. Product Code R202H).PLA-R848 conjugate having molecular weight of approximately 2500 Da andR848 content of approximately 13.6% by weight was synthesized by aring-opening process. PLA-PEG-Nicotine with a nicotine-terminated PEGblock of approximately 3,500 Da and DL-PLA block of approximately 15,000Da was synthesized. Polyvinyl alcohol (Mw=11,000-31,000, 87-89%hydrolyzed) was purchased from J. T. Baker (Part Number U232-08).

Methods for Nic,R848,OP-II Nanocarrier Production

Solutions were prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 69 mg/mL was prepared indistilled water at room temperature.

Solution 2: PLA-R848 @ 50 mg/mL, PLA @ 25 mg/mL, and PLA-PEG-Nicotine @25 mg/mL in dichloromethane was prepared by dissolving the polymers at100 mg/mL, combining the PLA-R848 and PLA solutions at a 2:1 ratio, andthen adding 1 part PLA-PEG-Nicotine solution to 3 parts of thePLA-R848/PLA solution.

Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in deionized water.

Solution 4: 70 mM phosphate buffer, pH 8.

A primary (W1/O) emulsion was first created using Solution 1 andSolution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were combined ina small glass pressure tube and sonicated at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary (W1/O/W2)emulsion was then formed by adding Solution 3 (2.0 mL) to the primaryemulsion and sonicating at 35% amplitude for 40 seconds using theBranson Digital Sonifier 250. The secondary emulsion was added to abeaker containing 70 mM phosphate buffer solution (30 mL) in an open 50ml beaker and stirred at room temperature for 2 hours to allow for thedichloromethane to evaporate and for the nanocarriers to form insuspension. A portion of the suspended nanocarriers were washed bytransferring the nanocarrier suspension to a centrifuge tube, spinningat 5,300 rcf for 60 minutes, removing the supernatant, and re-suspendingthe pellet in phosphate buffered saline. This washing procedure wasrepeated and then the pellet was re-suspended in phosphate bufferedsaline to achieve nanocarrier suspension having a nominal concentrationof 10 mg/mL on a polymer basis. The suspension was stored frozen at −20°C. until use.

TABLE 1 Nic, R848, OP-II Nanocarrier Characterization Effective TLRAgonist, T-cell helper Nanocarrier ID Diameter (nm) % w/w peptide, % w/wNic, R848, OP-II 234 R848, 0.7 Ova 323-339, 1.8

Materials for Nic,Ø,OP-II Nanocarrier Formulations

Ovalbumin peptide 323-339 amide TFA salt, was purchased from BachemAmericas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Part#4064565.) PLA with an inherent viscosity of 0.19 dL/g was purchasedfrom Boehringer Ingelheim (Ingelheim Germany. Product Code R202H).PLA-PEG-Nicotine with a nicotine-terminated PEG block of approximately3,500 Da and DL-PLA block of approximately 15,000 Da was synthesized.Polyvinyl alcohol (MW=11,000-31,000, 87-89% hydrolyzed) was purchasedfrom J. T. Baker (Part Number U232-08).

Methods for Nic,Ø,OP-II Nanocarrier Production

Solutions were prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 69 mg/mL was prepared in 0.13Nhydrochloric acid at room temperature.

Solution 2: PLA @ 75 mg/mL and PLA-PEG-Nicotine @ 25 mg/mL indichloromethane was prepared by dissolving PLA @ 100 mg/mL indichloromethane and PLA-PEG-Nicotine at 100 mg/mL in dichloromethane,then combining 3 parts of the PLA solution to 1 part of thePLA-PEG-Nicotine solution.

Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in deionized water.

Solution 4: 70 mM phosphate buffer, pH 8.

A primary (W1/O) emulsion was first created using Solution 1 andSolution 2. Solution 1 (0.1 mL) and Solution 2 (1.0 mL) were combined ina small glass pressure tube and sonicated at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary (W1/O/W2)emulsion was then formed by adding Solution 3 (2.0 mL) to the primaryemulsion and sonicating at 35% amplitude for 40 seconds using theBranson Digital Sonifier 250. The secondary emulsion was added to abeaker containing 70 mM phosphate buffer solution (30 mL) in an open 50ml beaker and stirred at room temperature for 2 hours to allow for thedichloromethane to evaporate and for the nanocarriers to form insuspension. A portion of the suspended nanocarriers were washed bytransferring the nanocarrier suspension to centrifuge tubes, spinning at5300 rcf for 60 minutes, removing the supernatant, and re-suspending thepellet in phosphate buffered saline. This washing procedure was repeatedand then the pellet was re-suspended in phosphate buffered saline toachieve nanocarrier suspension having a nominal concentration of 10mg/mL on a polymer basis. The suspension was stored frozen at −20° C.until use.

TABLE 2 Nanocarrier Characterization Effective TLR Agonist, T-cellhelper Nanocarrier ID Diameter (nm) % w/w peptide, % w/w Nic, Ø, OP-II248 None Ova, 2.2 (Ø = no adjuvant)

Results

Anti-nicotine antibody titers in mice immunized with NC containingsurface nicotine and T-helper peptide OP-II with or without R848 (5animals/group; s.c., 100 μg of NC per injection, 3 times with 4-wkintervals). Titers for days 26 and 40 after the 1^(st) immunization areshown (ELISA against polylysine-nicotine). Group 1: immunized withNP[Nic,R848,OP-II] (3.1% of NC-conjugated R848); group 2: immunized withNP[Nic,Ø,OP-II] (no R848 bound to NC) admixed with 20 μg of free R848.

These results demonstrate that conjugation of R848 to NC resulted in astronger adjuvant effect than utilization of free R848 admixed to NCthat does not contain R848. When identical amounts of two NCs, onecontaining surface nicotine, T-helper peptide OP-II and R848(NC[Nic,R848,OP-II]), and another containing the same ingredients, butwithout R848 (NC[Nic,Ø,OP-II]) were used for animal immunization, ahigher antibody response was observed for R848 that had been conjugatedto NC even if a substantially higher amount of free R848 (>6-fold) wasadmixed to NP[Nic,Ø,OP-II] prior to immunization compared to amount ofNC-conjugated R848 (FIG. 6).

Example 12 Nanocarriers with Entrapped Adjuvant Results in LowerSystemic Proinflammatory Cytokine Induction Materials for NanocarrierFormulations

Ovalbumin peptide 323-339 amide acetate salt, was purchased from BachemAmericas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Product code4065609.) PS-1826 DNA oligonucleotide with fully phosphorothioatedbackbone having nucleotide sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′(SEQ ID NO: 2) with a sodium counter-ion was purchased from Oligos Etc.(9775 SW Commerce Circle C-6, Wilsonville, Oreg. 97070.) PLA with aninherent viscosity of 0.19 dL/g was purchased from Boehringer Ingelheim(Ingelheim Germany. Product Code R202H). PLA-PEG-Nicotine with anicotine-terminated PEG block of approximately 5,000 Da and DL-PLA blockof approximately 17,000 Da was synthesized. Polyvinyl alcohol(Mw=11,000-31,000, 87-89% hydrolyzed) was purchased from J. T. Baker(Part Number U232-08).

Methods for Nanocarrier Production

Solutions were prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 70 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13N hydrochloric acid solution at room temperature.

Solution 2: 0.19-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml indichloromethane. The solution was prepared by separately dissolving PLA@ 100 mg/mL in dichloromethane and PLA-PEG-nicotine @ 100 mg/mL indichloromethane, then mixing the solutions by adding 3 parts PLAsolution for each part of PLA-PEG-nicotine solution.

Solution 3: Oligonucleotide (PS-1826) @ 200 mg/ml in purified water. Thesolution was prepared by dissolving oligonucleotide in purified water atroom temperature.

Solution 4: Same as solution 2.

Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

Two separate primary water in oil emulsions were prepared. W1/02 wasprepared by combining solution 1 (0.1 mL) and solution 2 (1.0 mL) in asmall pressure tube and sonicating at 50% amplitude for 40 seconds usinga Branson Digital Sonifier 250. W3/O4 was prepared by combining solution3 (0.1 mL) and solution 4 (1.0 mL) in a small pressure tube andsonicating at 50% amplitude for 40 seconds using a Branson DigitalSonifier 250. A third emulsion with two inner emulsion phases([W1/O2,W3/O4]/W5) emulsion was prepared by combining 0.5 ml of eachprimary emulsion (W1/O2 and W3/O4) and solution 5 (3.0 mL) andsonicating at 30% amplitude for 60 seconds using the Branson DigitalSonifier 250.

The third emulsion was added to an open 50 mL beaker containing 70 mM pH8 phosphate buffer solution (30 mL) and stirred at room temperature for2 hours to evaporate dichloromethane and to form nanocarriers in aqueoussuspension. A portion of the nanocarriers was washed by transferring thesuspension to a centrifuge tube and spinning at 13,800 g for one hour,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated and the pellet wasre-suspended in phosphate buffered saline for a final nanocarrierdispersion of about 10 mg/mL.

The amounts of oligonucleotide and peptide in the nanocarrier weredetermined by HPLC analysis. The total dry-nanocarrier mass per mL ofsuspension was determined by a gravimetric method and was adjusted to 5mg/mL. Particles were stored as refrigerated suspensions until use.

TABLE 3 Nanocarrier Characterization Effective TLR Agonist, T-cellhelper Nanocarrier Diameter (nm) % w/w peptide, % w/w 232 PS-1826, 6.4Ova, 2.2

Results

TNF-α and IL-6 were induced in sera of NC-CpG- and free CpG-inoculatedanimals. Animal groups were inoculated (s.c.) either with 100 μg ofNC-CpG (containing 5% CpG-1826) or with 5 μg of free CpG-1826. Atdifferent time-points post inoculation serum was collected from theanimals (3/group) by terminal bleed, pooled and assayed for cytokinepresence in ELISA (BD).

The results demonstrate that entrapment of adjuvant within NC results ina lower immediate systemic proinflammatory cytokine induction thanutilization of free adjuvant. When identical amounts of a CpG adjuvant,NC-entrapped or free, were used for inoculation, a substantially higherinduction of TNF-α and IL-6 in animal serum was observed for free CpGcompared to NC-entrapped CpG (FIG. 7).

Example 13 Nanocarriers with Entrapped Adjuvant Results in Similar orHigher Long-Term Systemic Induction of Immune Cytokines Materials forNanocarrier Formulations

Ovalbumin peptide 323-339 amide acetate salt, was purchased from BachemAmericas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Product code4065609.) PS-1826 DNA oligonucleotide with fully phosphorothioatedbackbone having nucleotide sequence 5′-TCC ATG ACG TTC CTG ACG TT-3′(SEQ ID NO: 2) with a sodium counter-ion was purchased from Oligos Etc.(9775 SW Commerce Circle C-6, Wilsonville, Oreg. 97070.) PLA with aninherent viscosity of 0.21 dL/g was purchased from SurModicsPharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211. ProductCode 100 DL 2A.) PLA-PEG-Nicotine with a nicotine-terminated PEG blockof approximately 5,000 Da and DL-PLA block of approximately 17,000 Dawas synthesized. Polyvinyl alcohol (MW=11,000-31,000, 87-89% hydrolyzed)was purchased from J. T. Baker (Part Number U232-08).

Methods for Nanocarrier Production

Solutions were prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 35 mg/mL in dilute hydrochloricacid aqueous solution. The solution was prepared by dissolving ovalbuminpeptide in 0.13N hydrochloric acid solution at room temperature.

Solution 2: 0.21-IV PLA @ 75 mg/mL and PLA-PEG-nicotine @ 25 mg/ml indichloromethane. The solution was prepared by separately dissolving PLA@ 100 mg/mL in dichloromethane and PLA-PEG-nicotine @ 100 mg/mL indichloromethane, then mixing the solutions by adding 3 parts PLAsolution for each part of PLA-PEG-nicotine solution.

Solution 3: Oligonucleotide (PS-1826) @ 200 mg/ml in purified water. Thesolution was prepared by dissolving oligonucleotide in purified water atroom temperature.

Solution 4: Same as Solution #2.

Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphatebuffer.

Two separate primary water in oil emulsions were prepared. W1/O2 wasprepared by combining solution 1 (0.2 mL) and solution 2 (1.0 mL) in asmall pressure tube and sonicating at 50% amplitude for 40 seconds usinga Branson Digital Sonifier 250. W3/O4 was prepared by combining solution3 (0.1 mL) and solution 4 (1.0 mL) in a small pressure tube andsonicating at 50% amplitude for 40 seconds using a Branson DigitalSonifier 250. A third emulsion with two inner emulsion([W1/O2,W3/O4]/W5) emulsion was prepared by combining 0.55 ml of eachprimary emulsion (W1/O2 and W3/O4) and solution 5 (3.0 mL) andsonicating at 30% amplitude for 60 seconds using the Branson DigitalSonifier 250.

The third emulsion was added to an open 50 mL beaker containing 70 mM pH8 phosphate buffer solution (30 mL) and stirred at room temperature for2 hours to evaporate dichloromethane and to form nanocarriers in aqueoussuspension. A portion of the nanocarriers was washed by transferring thesuspension to a centrifuge tube and spinning at 13,800 g for one hour,removing the supernatant, and re-suspending the pellet in phosphatebuffered saline. The washing procedure was repeated and the pellet wasre-suspended in phosphate buffered saline for a final nanocarrierdispersion of about 10 mg/mL.

The amounts of oligonucleotide and peptide in the nanocarrier weredetermined by HPLC analysis. The total dry-nanocarrier mass per mL ofsuspension was determined by a gravimetric method and was adjusted to 5mg/mL. Particles were stored as refrigerated suspensions until use.

TABLE 4 Nanocarrier Characterization Effective TLR Agonist, T-cellhelper Nanocarrier Diameter (nm) % w/w peptide, % w/w 217 PS-1826, 6.2Ova, Not Determined

Results

IFN-γ and IL-12 were induced in sera of NC-CpG- and free CpG-inoculatedanimals. Animal groups were inoculated (s.c.) with 100 μg of NC-CpG(containing 6% CpG-1826) or with 6 μg of free CpG-1826. At 24 hours postinoculation serum was collected from the animals (3/group) by terminalbleed, pooled and assayed for cytokine presence in ELISA (BD).

These results demonstrate that entrapment of adjuvant withinnanocarriers results in a similar or even higher long-term systemicinduction of immune cytokines compared to utilization of free adjuvant.When identical amounts of a CpG adjuvant, NC-entrapped or free, wereused for animal inoculation, a similar level of long-term induction ofsystemic IFN-γ and higher induction of IL-12 in animal serum wasobserved (FIG. 8).

1. A method comprising: providing a dose of adjuvant and a dose ofantigen, wherein at least a portion of the dose of adjuvant is coupledto synthetic nanocarriers, generating an antibody titer against theantigen through administration of the dose of adjuvant and the dose ofantigen to a subject, and choosing the dose of adjuvant to be less thana separate dose of adjuvant that results in an antibody titer similar tothat generated through administration of the dose of adjuvant and thedose of antigen to the subject. 2.-16. (canceled)
 17. The method ofclaim 1, wherein the synthetic nanocarriers comprise one or morepolymers.
 18. (canceled)
 19. The method of claim 17, wherein the one ormore polymers comprise or further comprise a polyester coupled to ahydrophilic polymer.
 20. (canceled)
 21. The method of claim 19, whereinthe hydrophilic polymer comprises a polyether.
 22. (canceled) 23.-25.(canceled)
 26. The method of claim 1, wherein the subject has cancer, aninfectious disease, a non-autoimmune metabolic disease, a degenerativedisease, an addiction, and atopic condition, asthma; chronic obstructivepulmonary disease (COPD) or a chronic infection.
 27. (canceled)
 28. Amethod comprising: providing a dose of adjuvant, wherein at least aportion of the dose of adjuvant is coupled to synthetic nanocarriers,generating a systemic cytokine release through administration of thedose of adjuvant to a subject, and choosing the dose of adjuvant to begreater than a separate dose of adjuvant that results in a systemiccytokine release similar to that generated through administration of thedose of adjuvant to the subject.
 29. The method of claim 28, wherein theadjuvant comprises an agonist for Toll-Like Receptors 3, 4, 5, 7, 8, or9 or a combination thereof. 30.-31. (canceled)
 32. The method of claim28, wherein the dose of adjuvant comprises two or more types ofadjuvants.
 33. The method of claim 28, wherein a portion of the dose ofadjuvant is not coupled to the synthetic nanocarriers.
 34. The method ofclaim 28, wherein more than one type of antigen are administered to thesubject.
 35. The method of claim 34, wherein at least a portion of adose of antigen(s) is coupled to the synthetic nanocarriers.
 36. Themethod of claim 34, wherein at least a portion of a dose of antigen(s)is not coupled to the synthetic nanocarriers.
 37. The method of claim34, wherein at least a portion of a dose of antigen(s) is coadministeredwith the synthetic nanocarriers.
 38. The method of claim 34, wherein atleast a portion of a dose of antigen(s) is not coadministered with thesynthetic nanocarriers. 39.-41. (cancelled)
 42. The method of claim 28,wherein the administration is by a route that comprises subcutaneous,intramuscular, intradermal, oral, intranasal, transmucosal, rectal;ophthalmic, transdermal or transcutaneous administration, or acombination thereof.
 43. The method of claim 28, wherein the syntheticnanocarriers comprise lipid nanoparticles, polymeric nanoparticles,metallic nanoparticles, surfactant-based emulsions, dendrimers,buckyballs, nanowires, virus-like particles, peptide or proteinparticles, nanoparticles that comprise a combination of nanomaterials,spheroidal nanoparticles, cuboidal nanoparticles, pyramidalnanoparticles, oblong nanoparticles, cylindrical nanoparticles, ortoroidal nanoparticles.
 44. The method of claim 28, wherein thesynthetic nanocarriers comprise one or more polymers.
 45. The method ofclaim 44, wherein the one or more polymers comprise a polyester. 46.-52.(canceled)
 53. The method of claim 28, wherein the subject has cancer,an infectious disease, a non-autoimmune metabolic disease, adegenerative disease, an addiction, and atopic condition, asthma;chronic obstructive pulmonary disease (COPD) or a chronic infection.54.-59. (canceled)
 60. A dose of adjuvant and dose of antigen as definedin claim 1, for use in therapy or prophylaxis. 61.-64. (canceled)